Broad-complex, but not Ecdysone receptor, is required for progression of the morphogenetic furrow in the Drosophila eye

Abstract

The progression of the morphogenetic furrow in the developing Drosophila eye is an early metamorphic, ecdysteroid-dependent event. Although Ecdysone receptor-encoded nuclear receptor isoforms are the only known ecdysteroid receptors, we show that the Ecdysone receptor gene is not required for furrow function. DHR78, which encodes another candidate ecdysteroid receptor, is also not required. In contrast, zinc finger-containing isoforms encoded by the early ecdysone response gene Broad-complex regulate furrow progression and photoreceptor specification. br-encoded Broad-complex subfunctions are required for furrow progression and proper R8 specification, and are antagonized by other subfunctions of Broad-complex. There is a switch from Broad complex Z2 to Z1 zinc-finger isoform expression at the furrow which requires Z2 expression and responds to Hedgehog signals. These results suggest that a novel hormone transduction hierarchy involving an uncharacterized receptor operates in the eye disc.

Full text

INTRODUCTION
Hormones play essential roles in the development of many
animals, particularly during postembryonic developmental
changes, such as amphibian and insect metamorphosis, and
mammalian puberty. While DNA-binding nuclear receptors
have been characterized for many steroid hormones, our
understanding of how steroid hormone signaling directs tissues
to differentiate remains rudimentary.
In Drosophila the ecdysteroids co-ordinate and trigger
the developmental events associated with molting and
metamorphosis. Ecdysone is released from the ring gland
in pulses during development, and converted to 20-
hydroxyecdysone (20-HE) in the fat body and target tissues
(Riddiford, 1993). Pulses of ecdysteroids at the end of larval
life and during pupal development direct many larval tissues
to die (Jiang et al., 1997), others to remodel (Levine et al.,
1995), and imaginal tissues to undergo proliferation,
morphogenesis and differentiation (Fristrom and Fristrom,
1993).
A heterodimeric nuclear receptor complex for 20-HE is
composed of the Ecdysone receptor (EcR) and Ultraspiracle
(Usp); the EcR/Usp dimer can bind 20-HE-responsive
regulatory DNA sequences, repressing transcription in the
absence of hormone, and activating it in the presence of 20-HE
(Cherbas et al., 1991; Koelle et al., 1991; Thomas et al., 1993;
Yao et al., 1993). Although null EcR or usp mutants die as
embryos or young larvae (Bender et al., 1997; Perrimon et al.,
1985), the essential roles of these genes at metamorphosis have
been demonstrated by rescuing mutants through early lethal
phases with inducible transgenes; both usp and EcR third instar
mutants fail to execute the normal program of metamorphosis
(Hall and Thummel, 1998; Li and Bender, 2000). Usp, an RXR
homolog, is thought to be able to dimerize with multiple steroid
receptors (Oro et al., 1990; Sutherland et al., 1995; Zelhof et
al., 1995a).
Ashburner and colleagues proposed that ecdysone signaling
directly activates a set of early puffs or genes. These early
genes were proposed to encode factors that transduce the
ecdysteroid signal and impose a temporal sequence of response
by activating other, late, genes as well as regulating their own
transcription (Ashburner et al., 1974). In support of this model,
the early gene Broad complex (BR-C; br – FlyBase) was found
to be required for the expression of late genes as well as for
metamorphic events in tissues other than the salivary glands
(Belyaeva et al., 1980; Belyaeva et al., 1981; Stewart et al.,
1972). The early genes at the E74, E75 and BR-C loci all
encode transcription factors, and have mutant phenotypes
consistent with their roles as transducers of ecdysone signals
(Burtis et al., 1990; Buszczak et al., 1999; DiBello et al., 1991;
Fletcher et al., 1995; Fletcher and Thummel, 1995; Kiss et al.,
1988; Segraves and Hogness, 1990).
The BR-C plays a key role in directing the appropriate,
stage-specific responses to hormone signaling at pupariation,
and may provide competence to cells to respond to ecdysone
signals (Karim et al., 1993). BR-C encodes a family of
1
Development 128, 1-11 (2001)
Printed in Great Britain © The Company of Biologists Limited 2001
DEV5416
The progression of the morphogenetic furrow in the
developing Drosophila eye is an early metamorphic,
ecdysteroid-dependent event. Although Ecdysone receptor-
encoded nuclear receptor isoforms are the only known
ecdysteroid receptors, we show that the Ecdysone receptor
gene is not required for furrow function. DHR78, which
encodes another candidate ecdysteroid receptor, is also
not required. In contrast, zinc finger-containing isoforms
encoded by the early ecdysone response gene Broad-
complex regulate furrow progression and photoreceptor
specification. br-encoded Broad-complex subfunctions are
required for furrow progression and proper R8
specification, and are antagonized by other subfunctions of
Broad-complex. There is a switch from Broad complex Z2
to Z1 zinc-finger isoform expression at the furrow which
requires Z2 expression and responds to Hedgehog signals.
These results suggest that a novel hormone transduction
hierarchy involving an uncharacterized receptor operates
in the eye disc.
Key words: Drosophila, Ecdysone, Morphogenetic furrow, BR-C,
EcR
SUMMARY
Broad-complex
, but not
Ecdysone receptor
, is required for progression of the
morphogenetic furrow in the
Drosophila
eye
Catherine A. Brennan1, Tong-Ruei Li2, Michael Bender2, Frank Hsiung3and Kevin Moses3,*
1Sloan-Kettering Institute, Box 193, 1275 York Avenue, New York, NY 10021, USA
2Department of Genetics, University of Georgia, Athens, GA 30602-7223, USA
3Department of Cell Biology, Emory University School of Medicine, 1648 Pierce Drive, Atlanta, GA 30322-3030, USA
*Author for correspondence (e-mail: kmoses@cellbio.emory.edu)
Accepted 27 October; published on WWW 27 November 2000

2
transcription factors that all possess one of four possible
alternative pairs of zinc fingers (Z1-Z4), and a BTB-POZ
protein interaction domain (Bardwell and Treisman, 1994;
Bayer et al., 1996a; DiBello et al., 1991; Zollman et al., 1994).
BR-C null mutants (non-pupariating; npr1, that lack all BR-C
protein isoforms) are unable to pupariate, and die after a
prolonged third instar, whereas mutants lacking isoform
subgroups die as pupae (Kiss et al., 1988; Stewart et al., 1972).
In mid and late third instar, activation of BR-C is less dependent
on EcR and Usp function than are E74 and E75 (Bender et al.,
1997; Hall and Thummel, 1998). In addition, BR-C function is
required for maximal induction of E74 and E75 (Belyaeva et
al., 1981; Karim et al., 1993; Zhimulev et al., 1982), whereas
BR-C is not reciprocally regulated by E74 (Fletcher and
Thummel, 1995).
There is evidence to support a novel hormone response
pathway in the mid-third instar. This includes the moderate
transcriptional induction of EcR, E74B and BR-C in a usp-
independent manner (Andres et al., 1993; Hall and Thummel,
1998; Huet et al., 1993; Thummel, 1996; von Kalm et al.,
1994). The DHR78 steroid receptor (Hr87 – FlyBase) may act
in this hierarchy; it is widely expressed at this time, and binds
to many early puffs (Fisk and Thummel, 1995; Fisk and
Thummel, 1998; Zelhof et al., 1995b). DHR78 mutants fail to
exhibit the characteristic mid-third instar pattern of hormone-
dependent gene activity. Ectopic expression of DHR78 is not
detrimental, suggesting that its activity may be regulated by a
ligand (Fisk and Thummel, 1998).
One dramatic consequence of ecdysteroid signaling at
metamorphosis is the transformation of the imaginal discs into
the epidermis and peripheral nervous system of the adult fly.
During pupal development, discs evert, elongate and terminally
differentiate in an ecdysteroid-dependent manner (Fristrom
and Fristrom, 1993). EcR, usp and BR-C mutants all show
defects in disc eversion and elongation (Fristrom et al., 1981;
Hall and Thummel, 1998; Kiss et al., 1988; Li and Bender,
2000).
Prior to overt morphogenesis, imaginal discs undergo
patterning and specification of neuronal elements (Cohen,
1993). For example, in the third instar eye disc, retinal
specification begins at the posterior margin and expands
anteriorly, with the addition of new rows of photoreceptor
clusters every two hours (Wolff and Ready, 1993). Associated
with the anterior boundary of retinal patterning are a
synchronized cell cycle arrest in G1 (Thomas et al., 1994;
Wolff and Ready, 1991), relaxation of heterochromatic gene
silencing (Lu et al., 1998), and a coordinated apical-basal
contraction of cells that produces the indentation known as the
morphogenetic furrow (Ready et al., 1976). The expression of
Hedgehog (Hh) and possibly Decapentaplegic (Dpp) signaling
proteins posterior to and in the furrow, respectively, drive the
furrow anteriorly at a controlled pace (Curtiss and Mlodzik,
2000; Heberlein and Treisman, 2000).
Such early neuronal differentiation in imaginal discs is also
under the control of ecdysteroids. Reduction of ecdysteroid
titer in the Drosophila eye disc in vivo with the ecdts mutation
results in irreversible furrow arrest and ommatidial disarray
(Brennan et al., 1998). 20-HE also sustains furrow progression
in vitro (Li and Meinertzhagen, 1995). The travelling furrow
is associated with the localized expression of a reporter of
ecdysteroid-dependent gene activity and of Broad-complex
proteins (Brennan et al., 1998). In the Manduca eye disc,
minimal levels of either ecdysone or 20-HE are required to
sustain furrow progression; below the threshold level, the
furrow reversibly stops during diapause (Champlin and
Truman, 1998b). In the Drosophila wing disc, the sensory
precursor cells in the wing margin undergo differentiation
and neurite outgrowth in response to 20-HE (Schubiger and
Truman, 2000). Accompanying the onset of differentiation in
imaginal discs is the relaxation of heterochromatic gene
silencing in imaginal tissues (Lu et al., 1998). In the eye disc,
this relaxation commences at the morphogenetic furrow, and in
wing, leg and eye imaginal discs, this relaxation can be induced
in vitro by 20-HE (Lu et al., 1998).
Recent evidence suggests that the molecular hierarchies that
transduce ecdysteroid signals in discs at these early stages are
distinct from those operating in larval tissues. In the wing disc,
usp represses BR-C during the mid third instar (Schubiger and
Truman, 2000); however, in the whole animal, no upregulation
of BR-C is seen at any time (Hall and Thummel, 1998). This
suggests that genetic interactions in imaginal discs may be
masked in studies of whole animals by effects in larval tissues,
which predominate by mass. The shared dependence among
imaginal discs of neural differentiation and heterochromatic
derepression on ecdysteroid signaling suggests that the eye disc
may be a good model for steroid hormone signaling in
differentiating tissues.
In this study we report whole disc and mosaic mutant
analysis to investigate the requirements for EcR, DHR78 and
BR-C during late third instar eye development. We show
that neither EcR nor DHR78 is required for normal furrow
progression or ommatidial assembly. In contrast, BR-C-
encoded alternative zinc finger isoforms are differentially
required in these processes. We also find evidence that the
pattern of BR-C expression is affected by Hh signaling.
MATERIALS AND METHODS
Drosophila
stocks and generation of clones
Clones were generated using EcRM554fs, a null allele (Bender et al.,
1997) by gamma-irradiation (1000R 137Cs) at 24-48 hours after egg-
laying. Disc clones were negatively marked with both arm-lacZ (at
51A) and EcR antibody stains, to confirm proximal mitotic
recombination and to control for perdurance of EcR protein. Adult
clones were negatively marked with a p(w+) transgene at 47A.
DHR782clones were also generated by gamma-irradiation, and
marked with arm-lacZ for disc clones, or a p(w+) transgene at 70ºC
for adult clones. Although DHR782is a null allele with a stop codon
in the ligand-binding domain (Fisk and Thummel, 1998), it produces
non-functional protein that is detected by anti-DHR78 antibody.
Therefore, clones could not be confirmed by loss of anti-DHR78
staining; however, in 12 clones examined, no ommatidial disruption
was seen. y w; EcRM554fs/EcRV559fs; hs-EcRB230.1/+ larvae were
rescued through the molt to the third instar by repeated heat shocks
and then maintained at 18°C for 4 more days. EcR protein is
undetectable 12 hours after heat shock (Li and Bender, 2000).
Hemizygous y npr13w, y br5w, y rbp5and y 2Bc1males were
identified by their yellow mouthhooks and dissected. BR-C
subfunction mutations used: npr13is null for all BR-C proteins, rbp5
disrupts Z1 isoforms, br5is null for Z2 isoforms and 2Bc1is null for
the Z3 isoforms (Bayer et al., 1997; Belyaeva et al., 1980; DiBello et
al., 1991; Emery et al., 1994; Kiss et al., 1988; Stewart et al., 1972).
BR-C, smo Mad, and Pka clones were generated by hsFlp-induced
C. A. Brennan and others

3Ecdysone signaling in eye development
FRT recombination (Xu and Rubin, 1993). FRT18 arm-lacZ; MKRS
hsFlp/TM6 males were crossed to y npr13w FRT18/FM7, y br5w
FRT18/FM7, y rbp5FRT18/FM7 and y 2Bc1FRT18/FM7 females.
smo2MadB1 FRT40/CyO and PKAh2 FRT40/CyO males were crossed
to w hsFlp; arm-lacZ FRT40/CyO females. Recombination was
induced by 1 hour heat shocks 24-48 hours after egg-laying.
Histochemistry
Eye discs were fixed and stained as previously described (Brennan et
al., 1998). Antibodies used: rabbit anti-β-galactosidase 1:2500
(CR7001RP2; Cortex Biochem); mouse anti-β-galactosidase 1:200
(Z378A, Promega); mouse anti-EcR 11D9.6 1:100 (Koelle et al.,
1991); rat anti-Elav 1:150 (Bier et al., 1988); mouse anti-BR-C core
25E9 1:100, anti-BR-C Z1 3C11 1:5 and mouse anti-BR-C Z3 9A7
1:5 (Emery et al., 1994); rabbit anti-Atonal 1:1500 (Jarman et al.,
1994); and mouse anti-Cyclin B 1:100 (Knoblich and Lehner, 1993).
All secondary antibodies were from Jackson Laboratories and
included: HRP-goat anti-rat IgG, rhodamine-donkey anti-rat, FITC
donkey anti-mouse, Cy5-donkey anti-rat, Cy5-donkey anti-mouse,
FITC-goat anti-mouse, TRITC-goat anti-mouse, Cy5-goat anti-
rabbit, FITC-goat anti-rabbit, and LRSC-goat anti-rabbit. In situ
hybridizations were performed as previously described (Mlodzik et
al., 1990; Tautz and Pfeifle, 1989). Digoxigenin-labelled antisense
RNA probes specific for Z1 and Z2 zinc finger-containing BR-C
isoforms were transcribed from EcoRI-linearized pSP64-Z1 and SalI-
linearized pSP65-Z2 (Bayer et al., 1996a).
RESULTS
Neither
EcR
nor
DHR78
is required for furrow
initiation or progression or ommatidial cluster
assembly
We examined mosaic clones and entire eye imaginal discs
lacking EcR function. EcR clones at the posterior margin of the
eye disc and in the center of the eye disc were normal or
showed very minor defects (Fig. 1A-F). This suggests that EcR
is not required for the initiation of retinal differentiation at
the posterior margin, for the anterior progression of the
furrow, or for proper ommatidial assembly. The mosaic
discs were stained with EcR antibody (Fig. 1B,E); lack of
staining in the clones demonstrated that perdurance of EcR
protein was not responsible for the lack of phenotype. We
note that the EcR antigen did not appear to be nuclear, as
one might expect. In these same specimens, the anti-Elav
antibody was capable of showing the nuclear localization
of Elav – thus it appears that the fixation and detergent
conditions in this experiment do allow full penetration of
the IgG primary and secondary antibodies. As details of the
EcR stain differ from the anti-β-galactosidase stain, it appears
that this stain is not simply bleed-through from the other
channel. Thus, the experiment does show a cytoplasmic
location for EcR in the eye disc. We have observed some
cytoplasmic localization of this antigen before, in the embryo
(Koelle et al., 1991), so this is not unprecedented.
Although six EcR clones both posterior and anterior (not
shown) to the furrow were examined, no clones spanning the
furrow were recovered. To confirm that EcR is not required
for furrow progression, and to test the possibility that EcR
may be non-autonomously required for early events in eye
development, we examined eye discs from EcR homozygotes
that had been rescued through the molt to the third instar by a
hs-EcR construct. Such animals show delayed wandering
behavior and pupariation failure, including the lack of anterior
spiracle eversion and larval cuticle hardening, and the
persistence of larval organs (Li and Bender, 2000). However,
in these discs, anterior progression of retinal differentiation
proceeded normally. Moreover, discs were seen in which the
furrow had traversed the entire eye field, reaching the antennal
boundary (Fig. 1G). This represents a late stage of eye disc
development that is normally only reached following
pupariation, and suggests that furrow progression is uncoupled
from metamorphic requirements for EcR function.
Initiation of furrow progression at the posterior margin
occurs prior to salivary chromosome puff stage 1 (Brennan et
al., 1998), suggesting that the ecdysteroid control of furrow
progression might be mediated by the alternative mid-third
instar hormone transduction hierarchy. To test whether
DHR78, which has been proposed to be the critical hormone
receptor at this early stage (Fisk and Thummel, 1998; Zelhof
et al., 1995b), mediates the ecdysteroid regulation of furrow
progression, we examined DHR78 mutant clones. However,
they displayed normal furrow progression and ommatidial
assembly (Fig. 1H,I).
Fig. 1. Neither EcR nor DHR78 is required for furrow
progression or ommatidial assembly. Third instar eye imaginal
discs. EcRM554fs clones in the center of the eye disc (A-C) and at
the posterior margin (D-F) are marked by loss of β-galactosidase
(A,D and green in C,F) and EcR (B,E) antibody stains. Staining
with an Elav antibody (red; C,F) reveals normal ommatidial
morphology. An eye disc null for EcR (see Material and
Methods) is stained with an Elav antibody (G) and shows normal
ommatidial morphology, as well as advancement of the furrow to
near the antennal disc boundary. A DHR782clone marked with
β-galactosidase (H; green in I) also shows normal ommatidial
morphology as seen with an Elav stain (red). Anterior is on the
right.

4
A BR-C isoform switch at the furrow
Despite this demonstrated lack of requirement for known or
candidate ecdysteroid receptors in transducing the hormone
requirement for furrow function, previous evidence suggested
that the early ecdysone response gene Broad-complex (BR-C)
plays a role in this process. Broad-complex proteins are
expressed at the furrow in an ecd-dependent manner, and discs
null for all BR-C function display defects in furrow progression
and ommatidial organization (Brennan et al., 1998).
We characterized the expression pattern in the eye disc of
the different zinc finger-containing BR-C isoforms using
antibody and mRNA probes (Fig. 2). An antibody that
recognizes all isoforms (‘core antibody’) stained cells both in
and flanking the furrow, with a peak of expression just posterior
to the furrow (Fig. 2A,B). BR-C is highly expressed in the
nuclei of cells in the peripodial membrane (Fig. 2C). BR-C
proteins appeared to be expressed in cells until they
differentiated; immediately posterior to the furrow, Elav-
expressing differentiating photoreceptors express BR-C,
whereas more posteriorly, expression is stronger in cone cells
(Fig. 2D). Staining with an antibody that detects only Z1-
containing isoforms of BR-C was restricted to posterior to
the furrow (Fig. 2E), and a Z3-specific antibody stained the
entire disc at very low levels, with slight upregulation in
photoreceptors posterior to the furrow (Fig. 2F). Although no
Z2-specific antibody is available, we reasoned that the staining
anterior to the furrow seen with the core antibody might be
represented by Z2-containing isoforms. This was confirmed by
in situ hybridization using probes specific for the Z1 and Z2
zinc fingers (Fig. 2G,H). Z2 mRNA was detected anterior to
the furrow, followed by Z1 posterior to the furrow. A switch in
BR-C isoforms expression from Z2 forms to Z1 forms around
the time of pupariation in imaginal discs has been described
(Emery et al., 1994). Our results show that this switch from
larval to prepupal forms is precocious and asynchronous in the
eye disc, and is associated with the morphogenetic furrow as
it traverses the disc. Although levels of BR-C expression
increase dramatically during the last few hours of the third
instar, corresponding to the late larval ecdysone pulse, BR-C
expression still remains highest near the furrow (see time series
in Fig. 2I-L).
npr1
and
br
are required for proper ommatidial
assembly
Bar is a dominant mutation causing premature arrest of the
furrow, which results in the deep anterior nick in the adult eye
(compare wild type in Fig. 3A with +/Bar in Fig. 3B; Kojima
et al., 1991). As Bar has the dominant effect of stopping the
furrow early, one might expect loss-of-function mutations at
other loci that normally act to promote furrow progression to
be genetic enhancers of Bar and loss-of-function mutations in
genes that normally antagonize the furrow to act as genetic
C. A. Brennan and others
Fig. 2. Broad-complex
expression in the eye disc. An
antibody that recognizes all
isoforms of Broad-complex
stains the eye disc anterior in,
and posterior to, the
morphogenetic furrow (A,B).
A and B are the same field; B
is a more basal optical section
to show staining in the furrow.
The same antibody stains
polytene nuclei in the
peripodial membrane (C).
Double staining of the disc
with the same antibody (green)
and Elav (red) in D shows that
while BR-C is initially
expressed in all cells in the
furrow, it remains strongly
expressed in photoreceptors as
they differentiate. More
posteriorlt, BR-C expression
has diminished in
photoreceptors, and is seen in
the newly formed, non-Elav
expressing cone cells (arrow
and circle in D). An antibody
that recognizes Z1-containing
isoforms of BR-C is restricted
to cells posterior to the furrow
(E), while an antibody that
recognizes Z3 isoforms lightly stains the whole disc, with slight increases in expression in newly formed photoreceptor cells (F). In situ
hybridization confirms that Z1 isoforms are expressed posterior to the furrow (G), and that the BR-C expression anterior to the furrow is
represented by Z2 isoforms (H). Furrows in G,H marked with arrowheads. (I-L) A time series of BR-C expression. Discs of a range of ages
were stained together with the common BR-C antibody and confocal images were collected under identical conditions. BR-C expression travels
with the furrow, and increases dramatically late in the third instar, corresponding to the time of the late larval ecdysteroid pulse. Anterior on the
right. Arrowheads show the position of the furrow.

5Ecdysone signaling in eye development
suppressors of Bar. Thus, we chose to examine genetic
interactions between BR-C sub loci and Bar.
We found that mutants defective for different BR-C
subfunctions displayed unexpected heterogeneity in their
genetic interactions with Bar, suggesting that the role of the
BR-C in the regulation of furrow function might be complex.
BR-C has several recessive lethal complementation groups that
correspond to mutations that remove the function of all or
individual zinc finger-containing isoforms subgroups. npr1
mutations lack all function, whereas rbp, br and 2Bc mutant
groups correspond to the loss of Z1-, Z2-, and Z3-containing
isoforms, respectively (see Table in Fig. 3; Bayer et al., 1997;
Crossgrove et al., 1996; DiBello et al., 1991; Emery et al.,
1994; Liu and Restifo, 1998; Sandstrom et al., 1997). Both
npr1/Bar and br/Bar eyes were significantly smaller than
+/Bar (Fig. 3C,D), indicating a dominant enhancement of the
Bar furrow-stop phenotype, consistent with the earlier reports
that the BR-C was required for furrow progression (Brennan et
al., 1998). However, br/Bar eyes were smaller than npr1/Bar,
suggesting that the BR-C might encode isoforms that act
antagonistically during furrow progression, so that the effect
of losing isoforms that positively regulate furrow progression
is more severe than losing all isoforms. This idea
is supported by the observation that rbp/Bar and
2Bc/Bar eyes are larger than +/Bar, suppressing
the phenotype, and possibly representing
furrow-antagonistic functions of rbp- or br-
encoded BR-C isoforms (Fig. 3E,F).
Hemizygous males of all BR-C mutant groups
survived through the third instar, and the eye
discs of these males displayed defects consistent
with the genetic interactions with Bar. npr1/Y
discs showed ommatidial disorganization, and
signs of furrow failure, including mature
ommatidial clusters at the furrow (Fig. 3G,
Brennan et al., 1998). br/Y discs showed a much
more dramatic failure of furrow progression, as
well as ommatidial disorganization (Fig. 3H).
rbp/Y and 2Bc/Y discs did not show any defects
(Fig. 3I,J). We note that while rbp does interact
strongly with Bar it shows no eye disc defect
alone – it may be that rbp is a redundant
function.
br
impairs furrow progression more
severely than
npr1
; both required for R8
patterning
Disc clones lacking individual BR-C
subfunctions were generated to further
characterize the mutant phenotypes (Fig. 4).
Both npr1 and br clones displayed defects in
ommatidial organization, including the wrong
number of photoreceptors in clusters (Fig.
4A,B). rbp and 2Bc clones appeared normal
(Fig. 4C,D).
To investigate the stages at which BR-C
function is required for normal third instar eye
development, we examined mosaic clones
spanning the morphogenetic furrow (Fig. 5). In
contrast to br clones, which were frequently
associated with some slowing of furrow
progression (Fig. 5B), none of over fifteen npr1 furrow-
spanning clones was associated with any retardation of the
furrow (Fig. 5A). This is consistent with the hemiygous disc
phenotypes that showed a more severe furrow effect in br than
npr1 mutants (Fig. 3). Neither npr1 (not shown) nor br (Fig.
5C) clones at the posterior margin of the disc showed any
visible defects in furrow initiation which is consistent with the
lack of requirement for ecd function for furrow initiation
(Brennan et al., 1998).
Both npr1 and br clones were associated with ommatidial
disarray, and were indistinguishable in this regard. Consistent
with this, both npr1 and br clones were defective in the
specification of the founding R8 photoreceptors, as shown by
Atonal expression (Fig. 5D-I). atonal is the proneural gene for
photoreceptor cells, and is first expressed in a broad
equivalence group of cells anterior to the furrow, and
subsequently restricted to expression in the R8 founder
photoreceptors (Jarman et al., 1994). A complex network of
inductive and inhibitory signals mainly involving the Notch
pathway specifies the R8 cells and ensures their correct spacing
(Baker et al., 1996; Cagan and Ready, 1989). In both npr1 and
br clones, clusters of three Atonal-positive R8 cells were seen.
Fig. 3. BR-C subfunctions differentially affect furrow function.
(Top left) Correspondence between the genetic subfunctions of BR-C and protein
isoforms; references in Materials and Methods. (A-F) Scanning electron micrographs
of eyes from females of the following genotypes: (A) wild-type; (B) +/FM7 B;
(C) npr13/FM7 B; (D) br5/FM7 B; (E) rbp5/FM7 B; (F) 2Bc1/FM7 B. br dominantly
enhances the Bar phenotype to a greater degree than does npr1; rbp and 2Bc both
appear to repress Bar. Note that A-F are all to the same scale. (G-J) Third instar eye
imaginal discs of males hemizygous for the same alleles of npr1, br, rbp and 2Bc,
stained for Elav. npr shows mild signs of furrow failure, while br discs are more
severely affected. rbp and 2Bc discs look normal. Anterior on the right.

6
This suggests a failure in the lateral inhibition mechanisms that
pattern these founder cells.
The cell cycle is influenced by steroid hormones, including
the insect ecdysteroids (Champlin and Truman, 1998a). Cell
cycle control in the Drosophila eye disc is essential for the
orderly specification of the retinal array; the synchronous cell
cycle arrest in G1 in the furrow is necessary for proper cell-cell
communication (Thomas et al., 1994; Wolff and Ready, 1993).
Dpp, produced from cells in the furrow, is thought to regulate
cell cycle synchronization in cells anterior to the furrow
(Horsfield et al., 1998; Pignoni and Zipursky, 1997). Cyclin B
is expressed in cells at the G2/M transition (Edgar and Lehner,
1996). Normally, Cyclin B is expressed in unsynchronized cells
far anterior to the furrow, turned off just anterior to and in the
furrow and reactivated in a tight band of cells posterior to the
furrow (Thomas et al., 1994; Wolff and Ready, 1991). We
assessed Cyclin B expression in br clones. In a long narrow br
clone that extends across the furrow, there was a delay in
cessation of Cyclin B expression anterior to the furrow (Fig. 5J-
L). However, close examination revealed that this was a non-
autonomous defect; the greatest delay occurred just adjacent to
the clone, in a region that is directly anterior to the section of
the mutant clone that crosses the furrow. We stained one other
br clone for Cyclin B that fell in this region of the eye disc and
it showed an indistinguishable phenotype. This suggests that the
delay in cell cycle synchronization anterior to the furrow is a
secondary defect resulting from delays in events at the furrow,
possibly including delayed production of Dpp.
BR-C isoform expression in mutant clones
br mutations lack the function of Z2 isoforms of the Broad-
complex, which are expressed in and anterior to the furrow in
the eye disc. This corresponds to the zone in which the earliest
defects in clones are noted: defects in R8 photoreceptor
patterning. To confirm the hypothesis that lack of Z2 isoform
expression anterior to the furrow is the cause of the defects in
br and npr1 clones, expression of various BR-C isoforms was
assessed in clones. As expected, in npr1 clones, expression of
all BR-C proteins was eliminated (data not shown). More
surprising was the finding that br clones were unreactive to an
antibody that recognizes all BR-C proteins (Fig. 6A). This
suggests that the expression of Z2 isoforms anterior to the
furrow is required for the subsequent expression of Z1 isoforms
posterior to the furrow. Evidence of such positive autoregulation
between Z2 and Z1 expression has been described (Bayer et al.,
1996b; Emery et al., 1994; Karim et al., 1993). rbp clones also
lacked expression of Z1 isoforms, as expected (Fig. 6C), yet
have no defects in retinal specification (compare Fig. 6B,D),
suggesting that the defects in br clones are due to the lack of
Z2 expression, and not to the lack of Z1. Although 2Bc clones
do show Z3 protein (data not shown), this protein is presumably
nonfunctional, as the 2Bc1allele has been shown to be a genetic
null (Emery et al., 1994).
Localized signals at the furrow and cellular
differentiation state influence BR-C expression
Although BR-C expression is dependent on highly diffusible
ecdysteroids, the pattern of BR-C expression in the eye disc is
spatially restricted. To test the possibility that local signals that
control the rate of furrow progression also influence the spatial
domain of BR-C expression, we examined clones in which cells
were unable to respond to the Hh and Dpp signals that drive
furrow progression, or in which the Hh signaling pathway
is constitutively activated (Curtiss and Mlodzik, 2000).
smoothened Mothers against dpp (smo Mad) double mutant
cells, which cannot transduce Hh or Dpp signals (Alcedo et al.,
1996; van den Heuvel and Ingham, 1996; Wiersdorff et al.,
1996), still expressed BR-C proteins near the furrow, showing
that reception of these signals is not strictly required for BR-C
expression (Fig. 7A-H). Aberrant nuclear migration patterns in
smo Mad cells distorted the appearance of the BR-C staining
pattern: basal optical sections showed that homozygous mutant
cells resembled their heterozygous neighbors in BR-C
expression levels (Fig. 7D). Neural differentiation was blocked
in smo Mad clones (Fig. 7B,F); BR-C expression persisted in
such clones far posterior to the furrow, suggesting a link between
differentiation and cessation of BR-C expression (Fig. 7G,H).
Constitutive activation of the Hh signaling pathway in Pka
mutant eye disc cells generates ectopic Mad-dependent circular
furrows radiating outwards (Pan and Rubin, 1995; Strutt et al.,
1995; Wiersdorff et al., 1996). A Pka clone examined just
posterior to the endogenous furrow showed ectopic expression
C. A. Brennan and others
Fig. 4. npr and br are required for proper ommatidial morphology.
Mutant clones for (A) npr13, (B) br5, (C) rbp5and (D) 2Bc1are
marked with β-galactosidase (left and green in merge on the right)
and stained with Elav antibody (middle panel and red in merge on
the right). Both npr1 and br clones display disrupted ommatidial
morphology, including clusters with too many and two few
photoreceptors. rbp and 2Bc clones show no defects. Anterior on the
right.

7Ecdysone signaling in eye development
of Z1 isoforms anterior to the endogenous Z1 domain,
surrounding the clone (Fig. 7I,J). This suggests that, although
expression of BR-C proteins at the furrow does not require Hh
or Dpp signaling, the timing of the switch from Z2 to Z1
isoforms is regulated, directly or indirectly, by these signals.
EcR
and
BR-C
but not
DHR78
are required for later
stages of eye development
To assess the requirements for BR-C, EcR and DHR78 for the
terminal differentiation that occurs during pupal
development, we examined white-marked clones
in the adult eye. br clones at the anterior margin
of the adult eye showed a small nick and subtle
disordering of the ommatidial array, defects that
probably result from the furrow-associated defects
described above (Fig. 8A). In contrast, npr1 clones
showed a scar, indicative of a requirement for non-
br-encoded BR-C functions in later stages of
maturation (Fig. 8B). Such defects often represent
failure of differentiation, and subsequent death of
the affected photoreceptors. EcR clones also
produced scars (Fig. 8C). This suggests that BR-C
might function downstream of EcR during later
stages of eye development, or that the two genes
function in parallel, regulating maturation events.
Either model would be consistent with the
demonstrated requirements for 20-HE for later
events in eye morphogenesis (Li and
Meinertzhagen, 1997). Homozygous loss-of-
function clones of the repressor dSin3A also leave
retinal scars (Neufeld et al., 1998), suggesting that
the repressive functions of EcR may be important
for proper morphogenesis. DHR78 clones in the
adult eye showed no defects (Fig. 8D).
DISCUSSION
We have shown that the ecdysteroid requirement
for furrow progression and orderly ommatidial
assembly in Drosophila eye development does not
depend on the activity of two known steroid
hormone receptors that are required for many of
the events of metamorphosis, EcR and DHR78.
Mutant EcR or DHR78 clones show no defects in
ommatidial specification, and eye discs from
larvae lacking detectable EcR protein display
normal furrow progression, although EcR is
required for later events in eye development.
In contrast, we found that BR-C proteins, whose
expression is lost in discs in which the ecdysteroid
titer is reduced in vivo (Brennan et al., 1998), play
important roles in third instar eye imaginal disc
development. We have shown that BR-C function
is not required for initiation of retinal patterning at
the posterior margin, but is required to maintain
furrow progression, for the appropriate
specification of founding R8 photoreceptors in the
furrow, for proper ommatidial assembly and for
later maturation events. These requirements for
BR-C are differentially met by distinct isoform-
encoding genetic subfunctions of the gene. The furrow-
promoting activity of BR-C is supplied by the br subfunction,
which encodes Z2 isoforms. A furrow-repressing activity
appears to be encoded by other BR-C subfunctions, as npr1
(BR-C null) discs and clones show less severe impairment of
furrow progression than br. We were not able to identify the
furrow-antagonizing genetic subfunction of npr1; the lack of
furrow acceleration in rbp (Z1-encoding) or 2Bc (Z3-encoding)
discs and clones may be due to redundancy with each other or
Fig. 5. br and npr functions are required for R8 specification, while only br
functions are strongly required for furrow advancement. While no furrow-spanning
npr clones were associated with retardation of neural specification (A), br clones
frequently showed furrow delay (B). Clones are marked in white outline, Elav in
red. Neither npr (not shown) nor br clones at the posterior margin (C) were
associated with failure of photoreceptor development or furrow initiation. All clones
were marked with lacZ, shown in green in C,F,I,L. npr and br clones were assayed
for Atonal expression (red in F,I) and Cyclin B (red in L). Both npr (D-F) and br
(G-I) clones showed defects in R8 specification, especially excess numbers of R8
cells in a cluster (arrowheads). A br clone (J-L) was associated with delays in
patterns of Cyclin B expression, both in the band of expression posterior to the
furrow and the cessation of expression anterior to the furrow. While the former is
autonomous, the latter (arrow) is non-autonomous and likely results from delays in
inductive events in the mutant tissue spanning the clone directly posterior. Anterior
on the right.

8
other ecdysone-response genes, or may suggest there are other
BR-C functions not represented by br, rbp or 2Bc. br and npr1
disc tissue was similarly impaired in proper R8 specification
and subsequent ommatidial organization. Both npr and br
tissue in the eye lack BR-C expression, as detected with the
core antibody; the reason for the more severe furrow phenotype
of br than npr is not known.
We found that a switch from Z2-containing to Z1-containing
BR-C isoform expression occurs at the morphogenetic furrow.
This isoform switch normally occurs in imaginal discs at
pupariation, suggesting that the furrow may mark a general
boundary between larval and prepupal patterns of gene
expression. We also found evidence that expression of Z2
isoforms anterior to the furrow may be required for subsequent
Z1 expression posterior to the furrow; br clones, whose
primary defect is failure of Z2 isoform expression, also failed
to express Z1 isoforms. The primary failure in br and npr1
discs appears to occur in or anterior to the furrow,
corresponding to the zone of Z2 expression, and is manifested
as failure to properly specify R8 cells. Our result showing
ectopic Z1 isoform expression near a Pka clone suggests that
the switch from Z2 to Z1 isoforms may be influenced by
furrow-associated expression of Hh and Dpp.
How is the ecdysteroid signal transduced during
furrow progression?
We have found that EcR does not mediate the ecdysteroid
requirement for morphogenetic furrow progression. The lack
of a requirement for EcR in furrow progression is surprising
for several reasons. EcR is expressed in the eye imaginal disc
during the time of furrow progression (Fig. 1), and is required
at this time in other tissues (Li and Bender, 2000). We had
previously found that a heptamer of Ecdysone Response
Element (EcRE) sequences was able to drive expression of a
reporter in the furrow (Brennan et al., 1998). Evidence
C. A. Brennan and others
Fig. 6. BR-C isoform expression in mutant clones. (A,B) br5clone
stained with antibody recognizing all forms of BR-C protein. Lack of
staining indicates br clones lack both Z2 and Z1 isoforms.
(C,D) rbp5clone stained with antibody recognizing Z1 isoforms of
BR-C, shows lack of Z1 expression in clone. Elav stain (red) shows
position of clone relative to furrow and indicates that lack of Z1
expression in br clone is not the source of ommatidial defects,
because the rbp clone also lacks Z1, yet shows no defects. Anterior
on the right.
Fig. 7. Hh and Dpp signaling and state of differentiation affect BR-C
expression. Approximate position of furrow indicated by yellow
lines. Anterior on right. (A-D) Furrow-spanning smo2MadB1 clones
(arrowheads) show neural differentiation failure (B) and aberrant
nuclear migration (compare apical and basal focal planes in C,D), but
no reduced expression of total BR-C protein. (E-H) Cells in posterior
smo2MadB1 clones (arrows) have failed to differentiate (F), and
show persistent BR-C staining. (I-J) PKAh2 clone has non-
autonomously induced precocious expression of BR-C Z1 isoforms
adjacent to clone (arrowheads).

9Ecdysone signaling in eye development
suggesting that only the EcR/Usp dimer is able to activate
transcription from EcREs includes that EcRE in Schneider
cells is bound by a single complex that is destroyed by anti-
EcR antibodies, EcR/Usp binds EcRE in the presence or
absence of hormone, and transfection of EcR confers 20-HE
responsiveness to cells carrying an EcRE reporter (Koelle et
al., 1991; Yao et al., 1992). The lack of EcR requirement for
furrow progression suggests either that a contribution by EcR
to furrow regulation is masked by redundant contributions by
another ecdysteroid receptor, or that an unidentified receptor
that can also activate transcription from EcREs exclusively
mediates the hormone regulation of furrow progression.
Redundancy seems unlikely because loss of EcR function has
strong phenotypes in almost all tissues where it is expected to
function, including at the larval molts and at pupariation (Li
and Bender, 2000). One candidate steroid receptor to mediate
the hormonal regulation of furrow progression is DHR96; it is
expressed during the mid-late third instar, and is able to bind
EcREs in gel-shift assays (Fisk and Thummel, 1995).
The EcR clone phenotypes in both discs and adult eyes are
distinct from those of usp, which are reported to produce
furrow acceleration in the disc and abnormal rhabdomeres, but
not scars, in the eye (Oro et al., 1992; Zelhof et al., 1995a).
These results, along with earlier findings of distinct embryonic
and early metamorphic phenotypes (Bender et al., 1997; Hall
and Thummel, 1998; Li and Bender, 2000; Perrimon et al.,
1985), suggest that EcR and Usp each have functions
independent of the other.
Does the receptor that transduces the ecdysteroid
requirement for furrow progression include Usp? usp retinal
clones are reported to show variable degrees of furrow
acceleration that correlate to some degree with stage of
development; clones spanning furrows near the posterior
margin show a more marked degree of furrow advancement
than those examined at later stages (Zelhof et al., 1997). The
ecdysteroid titer increases dramatically during the time that the
furrow traverses the eye disc; it is possible that Usp represses
furrow progression at the low titers prevailing near the time of
furrow initiation, but that this repression is relieved by
increasing hormone concentrations over the next several hours.
This would be somewhat analogous with the situation in the
wing disc, where Usp repression of neural development is
relieved by 20-HE (Schubiger and Truman, 2000); however,
Usp repression of furrow progression at low to moderate
hormone levels could only be partial, because furrow
progression does occur.
Regulation and function of
BR-C
during furrow
progression
We previously reported that BR-C expression in the eye disc
is dependent on ecd function, suggesting that it is regulated by
ecdysteroid titer (Brennan et al., 1998). While BR-C
expression in the eye disc is unlikely to be regulated by EcR,
Usp may play a role. BR-C and usp disc clone phenotypes are
opposite in that the former shows some furrow retardation
while the latter shows a slight acceleration (Zelhof et al., 1997).
Possible Usp repression of BR-C, or Usp regulation of BR-C
isoform ratio could explain these differences. BR-C and usp
eye clones also resemble each other in that both show
ommatidial disarray. This similarity underscores the
importance of orderly progression of the furrow for subsequent
events, and may reflect an essential hormonal input that
regulates timing of furrow progression (Brennan and Moses,
2000).
Local events appear to regulate BR-C expression at a
number of levels. Two regulatory inputs into the switch from
Z2 to Z1 expression at the furrow have been suggested: (1) the
lack of Z1 expression in br clones suggests that prior
expression of Z2 isoforms anterior to the furrow is required for
subsequent expression of Z1 isoforms; (2) the ectopic Z1
expression seen surrounding a PKA clone suggests that Hh-
dependent signaling at the furrow also promotes the switch to
Z1 isoforms. Cessation of BR-C expression in eye disc cells
correlates with cell-fate determination; photoreceptors
diminish expression levels earlier than cone cells, and failure
to differentiate in smo Mad cells is associated with prolonged
expression of BR-C proteins. BR-C proteins are thought to
confer competence to respond to ecdysteroid signals (Karim et
al., 1993); in the asynchronously developing eye disc, it may
be essential to spatially restrict such competence.
How might BR-C proteins exert their effects on eye
development? Although ecdysteroids regulate ommatidial
patterning at many stages of eye development in Manduca
(Champlin and Truman, 1998b), our evidence suggests that the
critical stage at which early eye development in Drosophila is
regulated by Broad-complex is just anterior to and in the
furrow. This corresponds to the site of Z2 expression and the
zone at which the earliest defects are detected, in R8
specification. No transcriptional targets of BR-C have been
identified in the eye; the aberrant specification of R8 founders
suggests that targets may include elements of the Notch
signaling pathway. BR-C regulation of such targets is likely to
be complex, with different isoforms acting antagonistically.
We thank the Iowa Developmental Studies Hybridoma Bank, Greg
Guild, David Hogness, Andrew Jarman, Patrick O’Farrell, David
Strutt, Carl Thummel, Jessica Treisman and Laurie von Kalm for
antibodies and flies; Lucy Cherbas, Nora Ghbeish, Margrit Schubiger,
Carl Thummel, Chih Cheng Tsai and Kevin White for sharing results
before publication; and Laurie von Kalm and Kathryn Anderson for
comments on the manuscript. This research was supported by an NSF
graduate fellowship to C. A. B. and NIH grants (GM53681) to M. B.
and (EY09299) to K. M.
Fig. 8. BR-C and EcR are required for later events
in eye development. white-marked clones of br5
(A), npr13(B), EcRM554fs (C) and DHR782(D).
Anterior nick in br clone likely reflects furrow
function, while scars in npr1 and EcR clones
probably represent pupal requirements. DHR78
clone shows no defect. Anterior on the right.

10
REFERENCES
Alcedo, J., Ayzenzon, M., Von Ohlen, T., Noll, M. and Hooper, J. E. (1996).
The Drosophila smoothened gene encodes a seven-pass membrane protein,
a putative receptor for the Hedgehog signal. Cell 86, 221-232.
Andres, A. J., Fletcher, J. C., Karim, F. D. and Thummel, C. S. (1993).
Molecular analysis of the initiation of insect metamorphosis: a comparative
study of Drosophila ecdysteroid-regulated transcription. Dev. Biol. 160,
388-404.
Ashburner, M., Chihara, C., Meltzer, P. and Richards, G. (1974). Temporal
control of puffing activity in polytene chromosomes. Cold Spring Harb.
Symp. Quant. Biol. 38, 655-662.
Baker, N. E., Yu, S. and Han, D. (1996). Evolution of proneural atonal
expression during distinct regulatory phases in the developing Drosophila
eye. Curr. Biol. 6, 1290-1301.
Bardwell, V. J. and Treisman, R. (1994). The POZ domain: a conserved
protein-protein interaction motif. Genes Dev. 8, 1664-1677.
Bayer, C. A., Holley, B. and Fristrom, J. W. (1996a). A switch in Broad-
Complex zinc-finger isoform expression is regulated posttranscriptionally
during the metamorphosis of Drosophila imaginal discs. Dev. Biol. 177, 1-
14.
Bayer, C. A., von Kalm, L. and Fristrom, J. W. (1996b). Gene regulation in
imaginal disc and salivary gland development during Drosophila
metamorphosis. In Metamorphosis: Postembryonic Reprogramming of Gene
Expression in Amphibian and Insect Cells, (ed. L. I. Gilbert, J. R. Tata and
B. G. Atkinson), pp. 321-361. San Diego: Academic Press.
Bayer, C. A., Von Kalm, L. and Fristrom, J. W. (1997). Relationships
between protein isoforms and genetic functions demonstrate functional
redundancy at the Broad-Complex during Drosophila metamorphosis. Dev.
Biol. 187, 267-282.
Belyaeva, E. S., Aizenzon, M. G., Semeshin, V. F., Kiss, I., Koezka, K.,
Baritcheva, E. M., Gorelova, T. D. and Zhimulev, I. F. (1980).
Cytogenetic analysis of the 2B3-4 to 2B11 region of the X-chromosome of
Drosophila melanogaster. I. Cytology of the region and mutant
complementation groups. Chromosoma 81, 281-306.
Belyaeva, E. S., Vlassova, I. E., Biyasheva, Z. M., Kakpakov, V. T.,
Richards, G. and Zhimulev, I. F. (1981). Cytogenetic analysis of the 2B3-
4 – 2B11 region of the X chromosome of Drosophila melanogaster ll.
changes in 20-OH ecdysone puffing caused by genetic defects of puff 2B5.
Chromosoma 84, 207-219.
Bender, M., Imam, F. B., Talbot, W. S., Ganetzky, B. and Hogness, D. S.
(1997). Drosophila ecdysone receptor mutations reveal functional
differences among receptor isoforms. Cell 91, 777-788.
Brennan, C. A., Ashburner, M. and Moses, K. (1998). Ecdysone pathway
is required for furrow progression in the developing Drosophila eye.
Development 125, 2653-2664.
Brennan, C. A. and Moses, K. (2000). Determination of Drosophila
photoreceptors: timing is everything. Cell. Mol. Life Sci. 57, 195-214.
Burtis, K. C., Thummel, C. S., Jones, C. W., Karim, F. D. and Hogness,
D. S. (1990). The Drosophila 74EF early puff contains E74, a complex
ecdysone-inducible gene that encodes two ets-related proteins. Cell 61, 85-
99.
Buszczak, M., Freeman, M. R., Carlson, J. R., Bender, M., Cooley, L. and
Segraves, W. A. (1999). Ecdysone response genes govern egg chamber
development during mid-oogenesis in Drosophila. Development 126, 4581-
4589.
Cagan, R. L. and Ready, D. F. (1989). Notch is required for successive cell
decisions in the developing Drosophila retina. Genes Dev. 3, 1099-1112.
Champlin, D. T. and Truman, J. W. (1998a). Ecdysteroid control of cell
proliferation during optic lobe neurogenesis in the moth Manduca sexta.
Development 125, 269-277.
Champlin, D. T. and Truman, J. W. (1998b). Ecdysteroids govern two phases
of eye development during metamorphosis of the moth, Manduca sexta.
Development 125, 2009-2018.
Cherbas, L., Lee, K. and Cherbas, P. (1991). Identification of ecdysone
response elements by analysis of the Drosophila Eip28/29 gene. Genes Dev.
5, 120-131.
Cohen, S. M. (1993). Chapter 13: Imaginal disc development. In The
Development of Drosophila melanogaster. Vol. 2 (ed. M. Bate and A.
Martinez-Arias), pp. 747-842. Cold Spring Harbor: Cold Spring Harbor
Laboratory Press.
Crossgrove, K., Bayer, C. A., Fristrom, J. W. and Guild, G. M. (1996). The
Drosophila Broad-Complex early gene directly regulates late gene transcription
during the ecdysone-induced puffing cascade. Dev. Biol. 180, 745-758.
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.
DiBello, P. R., Withers, D. A., Bayer, C. A., Fristrom, J. W. and Guild, G.
M. (1991). The Drosophila Broad-Complex encodes a family of related
proteins containing zinc fingers. Genetics 129, 385-397.
Edgar, B. A. and Lehner, C. F. (1996). Developmental control of cell cycle
regulators: a fly’s perspective. Science 274, 1646-1652.
Emery, I. F., Bedian, V. and Guild, G. M. (1994). Differential expression of
Broad-Complex transcription factors may forecast tissue-specific
developmental fates during Drosophila metamorphosis. Development 120,
3275-3287.
Fisk, G. J. and Thummel, C. S. (1995). Isolation, regulation, and DNA-
binding properties of three Drosophila nuclear hormone receptor
superfamily members. Proc. Natl. Acad. Sci. USA 92, 10604-10608.
Fisk, G. J. and Thummel, C. S. (1998). The DHR78 nuclear receptor is
required for ecdysteroid signaling during the onset of Drosophila
metamorphosis. Cell 93, 543-555.
Fletcher, J. C., Burtis, K. C., Hogness, D. S. and Thummel, C. S. (1995).
The Drosophila E74 gene is required for metamorphosis and plays a role in
the polytene chromosome puffing response to ecdysone. Development 121,
1455-1465.
Fletcher, J. C. and Thummel, C. S. (1995). The Drosophila E74 gene is
required for the proper stage- and tissue- specific transcription of ecdysone-
regulated genes at the onset of metamorphosis. Development 121, 1411-
1421.
Fristrom, D. and Fristrom, J. W. (1993). The metamorphic development of
the adult epidermis. In The Development of Drosophila melanogaster. Vol.
2 (ed. M. Bate and A. Martinez-Arias), pp. 843-897. Cold Spring Harbor:
Cold Spring Harbor Laboratory Press.
Fristrom, D. K., Fekete, E. and Fristrom, J. W. (1981). Imaginal disc
development in a nonpupariating lethal mutant in Drosophila melanogaster.
Wilhelm Roux’s Arch. Dev. Biol. 190, 11-21.
Hall, B. L. and Thummel, C. S. (1998). The RXR homolog Ultraspiracle is
an essential component of the Drosophila ecdysone receptor. Development
125, 4709-4717.
Heberlein, U. and Treisman, J. E. (2000). Early retinal development in
Drosophila. In Vertebrate Eye Development. Vol. 31 (ed. M. E. Fini), pp.
37-50. Springer-Verlag.
Horsfield, J., Penton, A., Secombe, J., Hoffmann, F. M. and Richardson,
H. (1998). decapentaplegic is required for arrest in G1 phase during
Drosophila eye development. Development 125, 5069-5078.
Huet, F., Ruiz, C. and Richards, G. (1993). Puffs and PCR: the in vivo
dynamics of early gene expression during ecdysone responses in
Drosophila. Development 118, 613-627.
Jarman, A. P., Grell, E. H., Ackerman, L., Jan, L. Y. and Jan, Y. N. (1994).
atonal is the proneural gene for Drosophila photoreceptors. Nature 369,
398-400.
Jiang, C., Baehrecke, E. H. and Thummel, C. S. (1997). Steroid regulated
programmed cell death during Drosophila metamorphosis. Development
124, 4673-4683.
Karim, F. D., Guild, G. M. and Thummel, C. S. (1993). The Drosophila
Broad-Complex plays a key role in controlling ecdysone-regulated gene
expression at the onset of metamorphosis. Development 118, 977-988.
Kiss, I., Beaton, A. H., Tardiff, J., Fristrom, D. and Fristrom, J. W. (1988).
Interactions and developmental effects of mutations in the Broad-Complex
of Drosophila melanogaster. Genetics 118, 247-259.
Knoblich, J. A. and Lehner, C. F. (1993). Synergistic action of Drosophila
cyclins A and B during the G2-M transition. EMBO J. 12, 65-74.
Koelle, M. R., Talbot, W. S., Segraves, W. A., Bender, M. T., Cherbas, P.
and Hogness, D. S. (1991). The Drosophila EcR gene encodes an ecdysone
receptor, a new member of the steroid receptor superfamily. Cell 67, 59-77.
Kojima, T., Ishimaru, S., Higashijima, S., Takayama, E., Akimaru, H.,
Sone, M., Emori, Y. and Saigo, K. (1991). Identification of a different-type
homeobox gene, BarH1, possibly causing Bar (B) and Om(1D) mutations
in Drosophila. Proc. Natl. Acad. Sci. USA 88, 4343-4347.
Levine, R. B., Morton, D. B. and Restifo, L. L. (1995). Remodeling of the
insect nervous system. Curr. Opin. Neurobiol. 5, 28-35.
Li, C. and Meinertzhagen, I. A. (1995). Conditions for the primary culture of
eye imaginal discs from Drosophila melanogaster. J. Neurobiol. 28, 363-380.
Li, C. and Meinertzhagen, I. A. (1997). The effects of 20-hydroxyecdysone
on the differentiation in vitro of cells from the eye imaginal disc from
Drosophila melanogaster. Invert. Neurosci. 3, 57-69.
Li, T. and Bender, M. (2000). A conditional rescue system reveals essential
C. A. Brennan and others

11Ecdysone signaling in eye development
functions for the Ecdysone receptor (EcR) gene during molting and
metamorphosis in Drosophila. Development 127, 2897-2905.
Liu, E. and Restifo, L. L. (1998). Identification of a Broad Complex-regulated
enhancer in the developing visual system of Drosophila. J. Neurobiol. 34,
253-270.
Lu, B. Y., Ma, J. and Eissenberg, J. C. (1998). Developmental regulation of
heterochromatin-mediated gene silencing in Drosophila. Development 125,
2223-2234.
Mlodzik, M., Hiromi, Y., Weber, U., Goodman, C. S. and Rubin, G. M.
(1990). The Drosophila seven-up gene, a member of the steroid receptor
gene superfamily, controls photoreceptor cell fates. Cell 60, 211-224.
Neufeld, T. P., Tang, A. H. and Rubin, G. M. (1998). A genetic screen to
identify components of thesina signaling pathway in Drosophila eye
development. Genetics 148, 277-286.
Oro, A. E., McKeown, M. and Evans, R. M. (1990). Relationship between
the product of the Drosophila ultraspiracle locus and the vertebrate retinoid
X receptor. Nature 347, 298-301.
Oro, A. E., McKeown, M. and Evans, R. M. (1992). The Drosophila retinoid
X receptor homolog ultraspiracle functions in both female reproduction and
eye morphogenesis. Development 115, 449-462.
Pan, D. and Rubin, G. M. (1995). cAMP-dependent protein kinase and
hedgehog act antagonistically in regulating decapentaplegic transcription in
Drosophila imaginal discs. Cell 80, 543-552.
Perrimon, N., Engstrom, L. and Mahowald, A. P. (1985). Developmental
genetics of the 2C-D region of the Drosophila X chromosome. Genetics 111,
23-41.
Pignoni, F. and Zipursky, S. L. (1997). Induction of Drosophila eye
development by Decapentaplegic. Development 124, 271-278.
Ready, D. F., Hanson, T. E. and Benzer, S. (1976). Development of the
Drosophila retina, a neurocrystalline lattice. Dev. Biol. 53, 217-240.
Riddiford, L. M. (1993). Hormones and Drosophila development. In The
Development of Drosophila melanogaster. Vol. 2 (ed. M. Bate and A.
Martinez-Arias), pp. 899-939. Cold Spring Harbor: Cold Spring Harbor
Laboratory Press.
Sandstrom, D. J., Bayer, C. A., Fristrom, J. W. and Restifo, L. L. (1997).
Broad-Complex transcription factors regulate thoracic muscle attachment in
Drosophila. Dev. Biol. 181, 168-185.
Schubiger, M. and Truman, J. W. (2000). The RXR ortholog Usp suppresses
early metamorphic processes in Drosophila in the absence of ecdysteroids.
Development 127, 1151-1159.
Segraves, W. A. and Hogness, D. S. (1990). The E75 ecdysone-inducible gene
responsible for the 75B early puff in Drosophila encodes two new members
of the steroid receptor superfamily. Genes Dev. 4, 204-219.
Stewart, M., Murphy, C. and Fristrom, J. W. (1972). Recovery and
preliminary characterization of X chromosome mutants affecting imaginal
discs of Drosophila melanogaster. Dev. Biol. 27, 71-83.
Strutt, D. I., Wiersdorff, V. and Mlodzik, M. (1995). Regulation of furrow
progression in the Drosophila eye by cAMP-dependent protein kinase A.
Nature 373, 705-709.
Sutherland, J. D., Kozlova, T., Tzertzinis, G. and Kafatos, F. C. (1995).
Drosophila Hormone Receptor 38: a second partner for Drosophila USP
suggests an unexpected role for nuclear receptors of the nerve growth factor-
induced protein B type. Proc. Natl. Acad. Sci. USA 92, 7966-7970.
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization
method for the localization of specific RNAs in Drosophila embryos reveals
translational control of the segmentation gene hunchback. Chromosoma 98,
81-85.
Thomas, B. J., Gunning, D. A., Cho, J. and Zipursky, S. L. (1994). Cell
cycle progression in the developing Drosophila eye: roughex encodes a
novel protein required for the establishment of G1. Cell 77, 1003-1014.
Thomas, H. E., Stunnenberg, H. G. and Stewart, A. F. (1993).
Heterodimerization of the Drosophila ecdysone receptor with retinoid X
receptor and ultraspiracle. Nature 362, 471-475.
Thummel, C. (1996). Flies on steroids – Drosophila metamorphosis and the
mechanisms of steroid hormone action. Trends Genet. 12, 306-310.
van den Heuvel, M. and Ingham, P. W. (1996). smoothened encodes a
receptor-like serpentine protein required for hedgehog signalling. Nature
382, 547-551.
von Kalm, L., Crossgrove, K., Von Seggern, D., Guild, G. M. and
Beckendorf, S. K. (1994). The Broad-Complex directly controls a tissue-
specific response to the steroid hormone ecdysone at the onset of Drosophila
metamorphosis. EMBO J. 13, 3505-3516.
Wiersdorff, V., Lecuit, T., Cohen, S. M. and Mlodzik, M. (1996). Mad acts
downstream of Dpp receptors, revealing a differential requirement for dpp
signaling in initiation and propagation of morphogenesis in the Drosophila
eye. Development 122, 2153-2162.
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.
Wolff, T. and Ready, D. F. (1993). Pattern formation in the Drosophila retina.
In The Development of Drosophila melanogaster. Vol. 2 (ed. M. Bate and
A. Martinez-Arias), pp. 1277-1325. Cold Spring Harbor: Cold Spring
Harbor Laboratory Press.
Xu, T. and Rubin, G. M. (1993). Analysis of genetic mosaics in developing
and adult Drosophila tissues. Development 117, 1223-1237.
Yao, T. P., Forman, B. M., Jiang, Z., Cherbas, L., Chen, J. D., McKeown,
M., Cherbas, P. and Evans, R. M. (1993). Functional ecdysone receptor
is the product of EcR and ultraspiracle genes. Nature 366, 476-479.
Yao, T. P., Segraves, W. A., Oro, A. E., McKeown, M. and Evans, R. M.
(1992). Drosophila ultraspiracle modulates ecdysone receptor function via
heterodimer formation. Cell 71, 63-72.
Zelhof, A. C., Ghbeish, N., Tsai, C., Evans, R. M. and McKeown, M.
(1997). A role for Ultraspiracle, the Drosophila RXR, in morphogenetic
furrow movement and photoreceptor cluster formation. Development 124,
2499-2506.
Zelhof, A. C., Yao, T.-P., Chen, J. D., Evans, R. M. and McKeown, M.
(1995a). Seven-up inhibits ultraspiracle-based signaling pathways in vitro
and in vivo. Mol. Cell. Biol. 15, 6736-6745.
Zelhof, A. C., Yao, T.-P., Evans, R. M. and McKeown, M. (1995b).
Identification and characterization of a Drosophila nuclear receptor with the
ability to inhibit the ecdysone response. Proc. Natl. Acad. Sci. USA 92,
10477-10482.
Zhimulev, I. F., Vlassova, I. E. and Belyaeva, E. S. (1982). Cytogenetic
analysis of the 2B3-4-2B11 region of the X chromosome of Drosophila
melanogaster. III. Puffing disturbance in salivary gland chromosomes of
homozygotes for mutation l(1)pp1t10. Chromosoma 85, 659-672.
Zollman, S., Godt, D., Prive, G. G., Couderc, J. L. and Laski, F. A. (1994).
The BTB domain, found primarily in zinc finger proteins, defines an
evolutionarily conserved family that includes several developmentally
regulated genes in Drosophila. Proc. Natl. Acad. Sci. USA 91, 10717-10721.