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© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 2801-2809 doi:10.1242/dev.125179
RESEARCH ARTICLE
Anteroposterior patterning of Drosophila ocelli requires an
anti-repressor mechanism within the hh pathway mediated
by the Six3 gene Optix
ABSTRACT
In addition to compound eyes, most insects possess a set of three
dorsal ocelli that develop at the vertices of a triangular cuticle patch,
forming the ocellar complex. The wingless and hedgehog signaling
pathways, together with the transcription factor encoded by
orthodenticle, are known to play major roles in the specification
and patterning of the ocellar complex. Specifically, hedgehog is
responsible for the choice between ocellus and cuticle fates within
the ocellar complex primordium. However, the interactions between
signals and transcription factors known to date do not fully explain
how this choice is controlled. We show that this binary choice
depends on dynamic changes in the domains of hedgehog
signaling. In this dynamics, the restricted expression of engrailed,
a hedgehog signaling target, is key because it defines a domain
within the complex where hedgehog transcription is maintained
while the pathway activity is blocked. We further show that
the Drosophila Six3, optix, is expressed in and required for the
development of the anterior ocellus specifically, limiting the ocellar
expression domain of en. This finding confirms previous genetic
evidence that the spatial allocation of the primordia of anterior and
posterior ocelli is differentially regulated, which may apply to the
patterning of the insect head in general.
KEY WORDS: Drosophila, Gene network, Head patterning, Ocelli,
Optix
INTRODUCTION
The dorsal adult head of Drosophila derives from the dorsalanterior region of the eye-antennal imaginal disc. In addition, this
disc gives rise to the remaining head capsule, the eyes, the antennae
and the maxillary palps (Younossi-Hartestein, 1993; Dominguez
Casares, 2005). The dorsal head is patterned by the dynamic
interplay between orthodenticle [otd; also known as ocelliless (oc)],
which encodes an Otx family transcription factor, and the wingless
(wg; the fly Wnt1 homolog) and hedgehog (hh) signaling pathways
(Friedrich, 2006). The result of this patterning is the allocation of the
dorsal head, which lies in between the eyes, into three territories
(from lateral to medial): orbital cuticle, frons and ocellar complex
(OCx) (Fig. 1A). The OCx comprises three small and structurally
simple eyes termed the ocelli that are located at the vertices of a
triangular patch of cuticle, the so-called interocellar cuticle, which
also harbors a set of stereotypical bristles (Fig. 1). Ocelli are
widespread in insects, where they play a number of roles, including
CABD (Andalusian Centre for Developmental Biology), CSIC-Universidad Pablo de
Olavide-Junta de Andalucı́a, Campus UPO, Ctra. Utrera km1, Sevilla 41013, Spain.
*Author for correspondence ([email protected])
Received 10 April 2015; Accepted 29 June 2015
flight stabilization and as movement detectors triggering the escape
response (Goodman, 1981; Mizunami, 1995).
Work in past years has aimed at defining the functional
relationships between wg, hh and otd during the process of dorsal
head patterning in the disc. This work has expanded our
understanding of the general mechanisms by which the conserved
Wnt and Hh signaling pathways interact (Royet and Finkelstein,
1997) and of the development and evolution of the eyes and dorsal
head of arthropods (Posnien et al., 2010, 2011; Samadi et al., 2015;
Schomburg et al., 2015), and has helped in establishing parallels
between head patterning across phyla. For instance, members of
both the Wnt and Otx gene families are involved in anterior head/
neural tube patterning in both invertebrates and vertebrates
(Lichtneckert and Reichert, 2005; Rhinn et al., 2005; Schinko
et al., 2008; Steinmetz et al., 2011; Fu et al., 2012).
The development of the OCx is a typical example of regional
specification, as defined by Davidson (2001), in which the OCx
progenitor field is further subdivided to give rise to the three ocelli
and the interocellar cuticle. One of the earliest steps during the
development of the Drosophila head is the initiation of otd
expression by wg (Royet and Finkelstein, 1996; Blanco et al., 2009).
In late embryos, all cells of the eye-antennal disc primordium, which
can be marked by the expression of eyeless (ey) (Quiring et al., 1994;
Czerny et al., 1999), express Otd (supplementary material Fig. S1).
During larval development Otd expression progressively disappears
from the disc, and is only maintained in its dorsal anterior region,
where wg is expressed (Royet and Finkelstein, 1997). Otd in turn is
required to activate hh transcription (Royet and Finkelstein, 1996).
This results, in the early third instar (L3) disc, in the coexpression of
wg, hh and otd in the prospective OCx region (Royet and
Finkelstein, 1996) (Fig. 1A,D). However, wg transcription is next
repressed in the prospective OCx to allow the development of the
ocelli and the interocellar cuticle and bristles; otherwise, these
structures fail to develop and are replaced by frons, a more lateral
type of cuticle (Royet and Finkelstein, 1996).
During mid and late L3, the OCx region becomes further
subdivided into three domains: the central domain transcribes hh and
becomes the interocellar cuticle (IOC) region, while two adjacent
domains express eyes absent (eya) and sine oculis (so), which
encodes a Six1/2 transcription factor, and will become the anterior
(a) and posterior ( p) ocelli (OC) (Blanco et al., 2010; Brockmann
et al., 2011) (Fig. 1B,D). The mechanism by which this aOC-IOCpOC pattern is controlled by hh has recently been investigated
(Aguilar-Hidalgo et al., 2013) and relies on the differential
activation by the Hh signaling pathway of two Hh target genes:
eya and the homeobox transcription factor engrailed (en). Hh first
activates eya throughout the OCx region; then, en is turned on in a
more restricted domain, which results in the attenuation of the Hh
signaling pathway and the concomitant loss of eya from these cells.
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Maria A. Domı́nguez-Cejudo and Fernando Casares*
RESEARCH ARTICLE
Development (2015) 142, 2801-2809 doi:10.1242/dev.125179
Fig. 1. Expression dynamics of wg, hh and otd
during the patterning of the Drosophila ocellar
complex region. (A,B) Prospective ocellar complex
(OCx) region (outlined by the dashed line) in mid
(A, mL3) and late (B, lL3) wg-GAL4>UAS-GFP; hh-Z
discs. Discs were stained with anti-β-galactosidase
(hh-Z) and anti-Otd. (A) Low magnification view of the
eye-antennal disc. a, antennal disc. (A′) Higher
magnification of the boxed region in A. Separate
channels are shown in A′ and B′. (C) X-Gal-stained
wg-Z adult head. The ocellar complex structures are
contained within the red dashed line triangle. wg-Z is
also expressed as a crescent surrounding the ocellar
cups (asterisks) in pigment cells, starting during
pupal stages (not shown). Its regulation has not been
addressed in this study. (D) Schematic
representation of relevant gene expression changes
in the prospective ocellar complex region in eL3 and
lL3 discs. ‘anterior’, ‘posterior’, ‘medial’ and ‘lateral’
define the axes. The medial region (boxed in red) will
give rise to the ocellar complex, whereas the more
lateral regions will contribute to more lateral head
fates. Otd+ indicates high Otd expression. aOC and
pOC, anterior and posterior ocelli; IOC, interocellar
cuticle; Orb, orbital cuticle; Fr, frons; Pt, ptillinum.
RESULTS
wg does not repress hh transcription but that of its targets
eya and en
Work from the Finkelstein and Gehring labs identified many of the
regulatory events between wg, hh and otd that result in the clearing
of wg transcription from the dorsal-medial eye disc region to
become the prospective OCx (Fig. 1D) (Royet and Finkelstein,
1995; Blanco et al., 2009). The regulatory interactions inferred from
both studies highlight the same principle: wg is required for the
specification of the dorsal head through the stepwise activation of
otd and hh; the latter then increases Otd levels only in the medial
region. Here, in a negative-feedback loop, high Otd represses wg
and the OCx region is specified. However, there are also important
differences between the two gene regulatory networks. Whereas
Blanco and co-workers proposed that the segregation of the wg and
hh expression domains is indirect, mediated through otd, Royet and
Finkelstein suggested a direct mutual repression between these two
signaling pathways. In particular, it was shown that removing wg
function during the second half of L3 resulted in an expansion of the
hh domain, pointing to wg as a hh repressor.
To test this point, we carried out the converse experiment: we
overactivated the wg pathway throughout the prospective OCx and
analyzed the effects on hh transcription, Hh pathway activity and the
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Hh pathway targets en and eya. To do so, we drove expression of a
constitutively active form of Armadillo (Arm*), the Drosophila
β-catenin and nuclear transducer of the canonical wg pathway
(Peifer et al., 1991), using the ocellar driver oc2-GAL4 (Blanco
et al., 2009) in a hh-Z background (Fig. 2). Adult oc2>Arm* flies
lack all OCx structures, which are replaced by frons (Fig. 2A), a
more lateral type of tissue that depends on wg (Royet and
Finkelstein, 1996). In oc2>Arm* discs, transcription of hh, as
monitored by the hh-Z reporter, is not repressed (Fig. 2B,D), nor is
the accumulation of the activator form of Cubitus interruptus (CiA),
which is an indicator of an active Hh pathway (Aza-Blanc et al.,
1997; Ohlmeyer and Kalderon, 1998) (Fig. 2C,E). On the contrary,
we detect that the hh-Z domain expands in oc2>Arm* OCx
(compare Fig. 2B,D). However, in this same genotype the two major
hh targets, eya and en, are repressed (Fig. 2F,G). Therefore, wg
signaling represses the outcome of the hh signaling pathway by
blocking the expression of its targets, not by affecting hh
transcription or its primary transduction. In addition, in this
experiment wg signaling overactivation expands the hh expression
domain. These results are thus in line with the Blanco et al. (2009)
model of indirect regulatory interactions between hh and wg during
OCx patterning.
hh is necessary for activity of the otd autoregulatory
enhancer
Blanco and co-workers found that otd activates its own transcription
(a property of the network that had not been noticed by Royet and
Finkelstein) and described the oc7 DNA fragment as the otd
autoregulatory enhancer (Blanco et al., 2009). Since high Otd levels
build up in the prospective OCx (Royet and Finkelstein, 1995) and
otd is regulated by Hh, we wondered whether these high levels
could be the result of the activation of oc7 by Hh. Our experiments
addressing this question reveal that oc7-Z is not active in early L3
discs, when Otd expression is homogeneous (Fig. 3A), but becomes
active in later discs in the region showing highest Otd levels
(Fig. 3B,C). When the Hh pathway is knocked down in the OCx
region of the disc by expressing a dominant-negative form of the Hh
co-receptor patched ( ptc) (oc2>ptcΔloop2, or HhKD), the
DEVELOPMENT
Thus, the central region, expressing en and devoid of eya, becomes
the IOC, whereas the remaining flanking eya-expressing domains
become the retina-producing OC (Aguilar-Hidalgo et al., 2013).
However, a central question that remains to be answered for a
comprehensive understanding of ocellar specification is how the
changes in hh signaling domains are regulated during development,
as this signaling morphogen plays a major role in controlling the
specification and patterning of the OCx structures. Here, we have
followed the regulatory steps that lead from the onset of hh
expression to the establishment of its final expression domain, and
define how these steps are interconnected in a gene regulatory
network. We find that two transcriptional repressors, encoded by en
and the Drosophila Six3/6 gene Optix, are key players in this
network.
Fig. 2. wg signaling represses the hh targets en and eya, not hh
transcription or signaling. (A) OCx expression of a constitutively active form
of the wg signaling transducer Armadillo (Arm*) in oc2>Arm* adults results in
loss of OCx structures (compare with Fig. 1C). (B-G) Prospective OCx regions
in lL3 discs are outlined. (B,C) The wild-type expression patterns of Eya and hh
(hh-Z) (B) and of En and the activator form of Ci (CiA) (C) are shown for
comparison. (D,F) oc2>Arm*; hh-Z disc stained for β-galactosidase (hh-Z; D)
and Eya (F). (E,G) oc2>Arm* disc stained for CiA and En. In oc2>Arm* discs
neither hh-Z transcription (D) nor high CiA levels (E) are lost. By contrast, the
expression of Eya (F) and En (G) is lost. The double-headed arrows (B,D) mark
the extent of the hh-Z domain. Disc images are shown at the same
magnification.
expression of oc7-Z is lost, coinciding with the loss of the highest
Otd levels (Fig. 3D). To test if Hh is sufficient to activate oc7, we
expressed Hh in more lateral head regions using the 86c11-GAL4
driver (supplementary material Fig. S2A). In 86c11>Hh discs new
patches of oc7-Z expression are detected, and these are associated
with new Eya-positive domains (Fig. 3E). In fact, adult 86c11>Hh
heads show supernumerary OC in lateral head regions associated
with an expansion of oc7-Z expression (supplementary material
Fig. S2B).
These results confirm the functionality of the oc7 autoregulatory
enhancer defined by Blanco et al. (2009) and establish a mechanism
by which Hh is responsible for increasing the levels of Otd, by
showing that Hh is required to turn on the otd autoregulatory
enhancer oc7.
The hh expression domain contracts during patterning of the
OCx region
Even with the modifications introduced in the experiments above,
the existing OCx patterning model could not explain the expression
changes that we observed for hh. Most conspicuously, the mutual
positive regulation between hh and otd should maintain hh
transcription in the whole prospective OCx region, where Otd
levels are highest, which, however, is not the case (see Figs 1 and 4).
One possibility that could explain the hh expression dynamics is that
hh-expressing cells, which initially span the whole dorsal region
extending anteriorly up to the antennal domain (Fig. 4A),
reorganize to form a central hh-expressing patch (Fig. 4B,C), thus
explaining the apparent expression loss in the anterior and posterior
Development (2015) 142, 2801-2809 doi:10.1242/dev.125179
Fig. 3. hh regulates the expression of the otd autoregulatory enhancer
oc7. OCx regions of oc7-Z imaginal discs. (A-C) Spatiotemporal dynamics of
the otd and oc7-Z expression patterns in early (eL3), mid (mL3) and late (lL3)
L3 discs, stained for Otd and β-galactosidase. oc7 is not active in eL3 (A). Its
expression starts in mL3 (B) and becomes stronger in lL3 (C, white arrow). In
parallel, the medialmost Otd expression also increases as development
proceeds (B,C). (D) Knockdown of the Hh signal in oc2>ptcΔloop2 (HhKD) in
lL3 discs results in loss of oc7-Z activity (open arrow). The peak levels of Otd
are not reached. (E) Overexpression of Hh in regions lateral to the OCx using
the 86c11-GAL4 driver (86c11>Hh6.1) and stained for β-galactosidase (oc7-Z)
and Eya. Ectopic patches of Eya and oc7-Z expression are induced in these
lateral regions (arrows). Merged and separate channels are shown for all
experiments. Low, medium and high Otd levels are marked by +, ++ and +++,
respectively. Scale bars mark relative magnification of panels A to D.
sides of the domain. Alternatively, the restriction of the hh domain
could be the result of an attenuation of hh transcription in the
prospective ocellar domains.
To clarify this, we followed the lineage of hh-expressing cells in
the dorsal eye disc and compared it with hh expression in late L3
discs using a hh-GAL4 reporter and the G-TRACE method (Evans
et al., 2009). In this experiment, the RFP marker reports current hh
transcription, while GFP is permanently expressed in all cells that
have expressed hh and their descendants (i.e. in the hh ‘lineage’). If
the final hh pattern were derived from a rearrangement of cells, we
would not expect a major discrepancy between RFP (current) and
GFP (lineage) signals. However, if some cells had lost hh
transcription during development, some GFP cells would no
longer express RFP. The latter is what we observed (Fig. 4D;
n=8). Therefore, we conclude that the refinement of the hh pattern
during L3 requires the loss or repression of hh transcription in the
anterior and posterior regions of the OCx. It is in these regions that
eya remains active to specify the aOC and pOC.
en is required for hh maintenance in the IOC
During L3, hh transcription is lost from the anterior and posterior
regions of the OCx (Fig. 4) to become restricted to the prospective
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Fig. 4. Alterations in the hh expression domain are the result of changes
in hh transcription. (A-C) early (A) and late (B) hh-Z eye antennal discs,
stained for β-galactosidase (hh-Z). The disc in B has also been stained for Eya.
The double-headed arrow indicates the approximate extent of the OCx region.
The prospective OCx region (boxed in B) is enlarged in C. The hh-Z domain is
flanked by the OC Eya patches. (D) G-TRACE pattern of the hh-GAL4 line.
The current (red) and lineage (green) expression patterns are shown (merged
and individual channels). The arrows point to regions within the hh-GAL4
lineage where hh-GAL4 transcription has been lost.
IOC. In this region, the expression domain of hh is almost
coincident with that of en (Royet and Finkelstein, 1997; AguilarHidalgo et al., 2013). Previous work has shown that en acts as a hh
activator during embryo segmentation and in wing discs (Lee et al.,
1992; Mohler and Vani, 1992; Simmonds et al., 1995; Tabata et al.,
1995). However, during the development of the OCx, the initiation
of en expression is triggered by hh, placing en downstream of hh
during early ocellar development (Aguilar-Hidalgo et al., 2013).
Development (2015) 142, 2801-2809 doi:10.1242/dev.125179
These observations suggested a role for en not in the initiation, but
in the maintenance, of hh expression.
To test this, we first carried out two loss-of-function
experiments. In the first, we drove an enRNAi construct in a hhGAL4; UAS-GFP (hh>GFP) background. We reasoned that if hh
transcription required en, then knocking down en in this
background should result in the reduction of the hh-driven GFP
signal. Consistent with this, we found that in hh>GFP+enRNAi
discs the GFP signal was weaker than in hh>GFP+lacZ discs (in
this genotype an additional UAS transgene, UAS-lacZ, was
introduced to equalize the number of UAS transgenes in both
genotypes; Fig. 5A,B). We also noted a reduction in the size of the
eya-expressing patches, which is further compatible with weaker
Hh signaling, as eya is a hh target (Blanco et al., 2009; AguilarHidalgo et al., 2013). Moreover, hh>GFP+enRNAi adult flies
showed an almost complete replacement of the IOC by OC
(Fig. 5C), a phenotype similar to that previously described for en
null clones (Aguilar-Hidalgo et al., 2013). This confirmed the
efficacy of the RNAi-mediated en knockdown.
In the second experiment, we induced en null mutant clones in a
hh-Z background. In these clones the expression of hh-Z was
severely reduced (Fig. 5D). We then carried out the converse
experiment: to induce the expression of en in GFP-marked cell
clones in a hh-Z background. In this experiment, en-expressing
clones, located in regions adjacent to the normal ocellar hh
expression domain, activated the hh-Z reporter cell-autonomously
(Fig. 5E,E′). Thus, the results from both loss- and gain-of-function
manipulations of en expression supported the hypothesis that en
feeds back onto hh to maintain the characteristic high hh expression
levels in the IOC.
Optix is asymmetrically expressed within the OCx and
required for the development of the aOC
Having defined en as a positive regulator of hh in the OCx, we next
asked which mechanisms in the OCx prevent en from being more
widely expressed. Wider en expression would be expected since en is
a hh target and hh expression extends, initially, throughout the whole
prospective OCx. However, as en is a hh pathway repressor, wider
expression of en would result in the abrogation of OC formation.
This reasoning led us to hypothesize the existence of additional
genes with asymmetrical expression within the OCx region, which
would restrict the region within the OCx where en could be induced.
Fig. 5. en is necessary and sufficient to maintain high
hh transcription levels. (A,B) OCx regions of UASlacZ;; hh-GAL4,UAS-GFP (hh>GFP; A) and hh-GAL4,
UAS-GFP/UAS-enRNAi (hh>GFP+enRNAi; B), stained
with anti-Eya. GFP signal is reduced in hh>GFP
+enRNAi (B) relative to hh>GFP (A). In addition, in the
presence of enRNAi the Eya signal weakens and the gap
between the two Eya patches is partially filled with
Eya-expressing cells. (C) hh>enRNAi adult head. In
these flies, the IOC region is almost completely absent
(one IOC bristle remains) and only one large ocellus
forms. (D) en mutant clones (Df en) in the ocellar region,
marked by the absence of GFP, induced in a hh-Z
background. When these clones fall in the IOC region
(arrow), they lose hh-Z signal. (E) Flip-out en-expressing
clones (>>En, marked with GFP) in the dorsal anterior
region of the eye-antennal disc, corresponding to the
boxed region of the hh-Z disc shown in E′. The confocal
plane shown in E′ shows the endogenous hh-Z
expression domain (within the boxed region). The
confocal plane in E is more medial. GFP-marked enexpressing clones (E, arrows) activate hh-Z expression.
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Development (2015) 142, 2801-2809 doi:10.1242/dev.125179
In the eye-antennal disc, the Six3/6 homolog Optix has
been shown to be expressed in the undifferentiated cell population
of the developing eye (Seimiya and Gehring, 2000; Kenyon et al.,
2005). In this context, Optix is required to promote retinal
differentiation (Li et al., 2013). Interestingly, Optix is also
expressed in the prospective dorsal head of the eye-antennal disc
in a single domain (Seimiya and Gehring, 2000; Kenyon et al.,
2005). We examined expression using an Optix-specific
antibody, and found it to be expressed in a single anterior domain
that overlaps with the aOC Eya domain but not with the IOC region
(Fig. 6A) and that falls entirely within the broader otd domain
(supplementary material Fig. S3). To investigate the function
of Optix in this region, we used UAS-dsRNAi specific to Optix
driven by oc2-GAL4 (oc2>OptixRNAi or OptixKD), which
strongly reduced the Optix protein signal. Concomitantly,
the anterior domain of Eya was variably reduced or absent
(Fig. 6A,B). In oc2>OptixRNAi adult flies, the anterior ocellus
was frequently unfused, small or absent, again with variable
expressivity, whereas other ocellar structures, including the
posterior ocellus, remained unaffected (Fig. 6C,D). By contrast,
when we overexpressed Optix in the prospective IOC using a
hh-GAL4 driver (Hh>Optix), the IOC was reduced, as expected
from en repression (Fig. 6C,E).
To map more precisely the region of the adult head that derives
from this Optix-expressing domain, we drove expression of
UAS-lacZ with the OptixNP2631-GAL4 line, which is a GAL4
insertion in the vicinity of the Optix gene (see Materials and
Methods) that recapitulates Optix expression in the dorsal eye disc
(not shown). X-Gal-stained adult heads showed expression in a
medial stripe running from the anterior edge of the dorsal head
capsule to the aOC, but not beyond (Fig. 6C), in agreement with the
requirement for Optix in the aOC only. Therefore, the anterior
ocellus primordium, but not the posterior ocellus primordium, falls
within the medial-anterior dorsal head domain marked by Optix,
and requires Optix for its development.
Optix acts as an en repressor in the aOC primordium
The fact that the pOC develops without the need of Optix made us
think that, rather than acting as a positive factor, Optix could be
working to alleviate an ocellus fate repressor. Two pieces of
evidence suggested that en could be such a repressor. First, the
expression patterns of Optix and en were almost mutually exclusive
(Fig. 7A). Second, the gain of Optix function phenocopied en loss
(Fig. 6D) (Aguilar-Hidalgo et al., 2013).
To test whether Optix is required to set the anterior en expression
limit, we knocked down Optix expression by driving OptixRNAi in
the whole OCx region (using oc2-GAL4) or specifically in the
anterior ocellus (with NP2631-GAL4) and assayed their effects on
the expression of the en reporter en-Z. In discs of either genotype
(oc2>OptixRNAi or NP2631>OptixRNAi) we still detected low
levels of Eya expression in the aOC (see above and Fig. 7A-C),
which was likely to be due to residual Optix function. Accordingly,
the aOC was often reduced in oc2>OptixRNAi (Fig. 7B′; see also
Fig. 6B) or incorrectly fused along the dorsal midline in
NP2631>OptixRNAi (Fig. 7C′). When we checked the expression
of en-Z in oc2>OptixRNAi discs we observed an anterior expansion
of en-Z along the lateral regions of the OCx (Fig. 7B), which
corresponded in the adult head to a lateral-anterior extension of the
en-Z domain, surrounding the vestigial aOC (Fig. 7B′). When
OptixRNAi was driven by NP2631-GAL4, en-Z also expanded
anteriorly, but this time preferentially along the medial region of the
OCx. Accordingly, in the resulting adults, en-Z signal spread
anteriorly in the cuticle that splits the unfused aOC (Fig. 7C′).
Next, we overexpressed Optix in the prospective IOC domain
using a hh-GAL4 driver in an en-Z background. In these
experiments, en-Z expression was attenuated in the prospective
IOC, although not completely absent (Fig. 7D). This level of
reduction, however, seemed sufficient to block en action, as now the
expression of eya extended in between the aOC and pOC patches,
forming a continuous domain (Fig. 7D). In the adult, this resulted in
the reduction of the IOC fate (Fig. 6E), which depends on en
(Aguilar-Hidalgo et al., 2013). However, the size of the OC was not
increased, suggesting that the overexpression of Optix in the IOC
does not allow for expansion of OC fate, even when it now expresses
eya (Fig. 7D). Therefore, the finding that reduction of Optix results
in the anterior expansion of en, which impedes the activation of eya
by Hh signaling, would explain the loss or reduction of the aOC in
Optix knockdowns. In addition, the capacity of en to repress Optix
suggests that the border between the Optix and en domains might be
refined by mutual repression.
DISCUSSION
The relative simplicity of the OCx makes it an ideal system with
which to study in detail the mechanisms involved in the
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DEVELOPMENT
Fig. 6. Optix is expressed in the OCx overlapping the aOC Eya
domain and is required for normal development of the aOC.
(A,B) Prospective OCx regions in lL3 discs stained with anti-Optix
(red) and anti-Eya (green). Expression overlap is seen as yellow. In
oc2>OptixRNAi discs (B, OptixKD), Optix signal is reduced to
background levels [compare the regions marked with the red
arrows in A (control) and B]. In these discs, Eya expression in the
anterior patch is strongly reduced. (C-E) Adult heads of the
following genotypes: OptixNP2631>lacZ (C) stained with X-Gal to
detect β-galactosidase activity; (D) oc2>OptixRNAi, in which the
anterior ocellus is vestigial; (E) a hh>optix head, in which the IOC is
reduced. Note the absence of IOC bristles. The dashed lines (C,D)
mark the approximate anterior limit of the IOC.
RESEARCH ARTICLE
Development (2015) 142, 2801-2809 doi:10.1242/dev.125179
specification and patterning of a visual structure. Previous work had
described the functional relationships between wg, hh and otd
during the specification of the dorsal head, the region where the
OCx forms. The outcome of these interactions, a wg-cleared OCx
region, allows the subsequent specification of the ocellar structures,
namely the three ocelli (an eya/so-dependent structure) and the
intervening interocellar cuticle (an en-dependent structure), by the
Hh signaling pathway. However, it is not clear how this aOC-IOCpOC pattern is generated. More specifically, since this pattern
depends on hh, the question is to understand how hh controls
alternative fate decisions in this region. In this work, we have shown
that the hh signaling domain changes during this process and that
this change is essential for proper ocellar development. This
dynamics depends on the establishment of a feedback loop with the
hh signaling pathway target en, which, in turn, is restricted in its
expression domain by the action of the Drosophila Six3/6 homolog
Optix.
During the first half of L3 two Hh-related events occur. First, wg
transcription clears from the prospective ocellar region. This is
mediated by high Otd levels (Royet and Finkelstein, 1995), which
are achieved through the activation of an otd autoregulatory
enhancer [oc7 (Blanco et al., 2009)] by Hh signaling (Fig. 3).
Second, and parallel to the wg clearing, low levels of eya expression
are induced throughout the whole OCx (Aguilar-Hidalgo et al.,
2013). During the second half of L3, though, the initially uniform
expression domain of hh fades away to become restricted to its
central region, associated with the activation of en. This central
domain becomes the non-retinal interocellar cuticle (IOC), where en
represses the transduction of the hh signal. This change in hh
expression pattern, which defines the aOC-IOC-pOC domain
organization in the disc and the structure of the adult OCx, occurs
through transcriptional changes (Fig. 4). In particular, the
maintenance of hh in the central domain depends on en.
Therefore, after en expression is turned on by hh signaling
(Aguilar-Hidalgo et al., 2013), en feeds back positively on hh
transcription to maintain high hh expression levels (Fig. 5). In the
prospective IOC, the expression of en represses hh signaling
transduction and the initial expression of eya is lost. In the adjacent
regions, though, eya expression is maintained at high levels through
an autoregulatory loop that involves so (Brockmann et al., 2011). In
addition, a potential non-autonomous contribution of Hh, produced
2806
at the IOC region, cannot be excluded. The en-to-hh maintenance
function that we describe in the OCx resembles the well-established
role of en as a hh transcriptional activator in other contexts, such as
the embryo segmental stripes and the posterior compartment of the
wing disc (Mohler and Vani, 1992; Tabata et al., 1992, 1995; Zecca
et al., 1995), and could constitute a regulatory module that is
deployed in several developmental contexts, such as the developing
head.
We have further demonstrated that the peripheral reduction of the
hh domain is due to transcriptional regulation rather than cellular
rearrangements. This raises the question of how the reduction of hh
transcription outside the IOC region occurs. The most likely
possibility is that a hh activator is lost as the development of the
OCx region progresses through L3. We noted that hyperactivation of
the wg canonical pathway in the OCx region results in an expansion
of the hh-Z domain (Fig. 2). If wg were required to activate hh, the
indirect negative-feedback loop that results in the wg clearing from
the OCx region would also result in the loss of its activating action
on hh and the loss of hh transcription. This would be prevented only
in places where en was expressed. This hypothesis (wg being
required for hh expression in the OCx) is supported by the fact that,
in the embryonic head, wg activates hh expression (Bejsovec and
Martinez Arias, 1991; Lee et al., 1992). Nevertheless, it seems
contradictory to previous reports in which, using a temperaturesensitive wg allelic combination, the reduction of wg signaling
activity resulted in an enlargement of the OC and the IOC
(Royet and Finkelstein, 1996). However, this result could be
reconciled with wg acting as a hh activator. Early during L3, wg
would activate hh transcription (Fig. 2) while simultaneously
preventing the expression of en and eya, two targets of hh (Blanco
et al., 2009) (Fig. 2). Later in L3, high levels of Otd, produced after
the activation of the oc7 enhancer, result in the transcriptional
repression of wg in the prospective OCx region. This repressive
step leaves the wg expression domain restricted to more lateral
regions, where the frons and the orbital cuticle will be specified.
Removing wg function during this late period, as in the Royet and
Finkelstein (1996) experiment, would allow the activation of
Hh targets eya and en in a broader domain due to the nonautonomous action of secreted Hh. Since en maintains hh
transcription at high levels, late removal of wg should result in an
enlarged hh expression domain and an increase in the overall size of
DEVELOPMENT
Fig. 7. Optix regulates en expression. (A-C′) Late L3 (A-C) or
adult (A′-C′) OCx region of control (wt), oc2>OptixRNAi
(oc2>OptixRI) and NP2631>OptixRNAi (NP2631>OptixRI)
en-Z individuals. In discs, the attenuation of Optix in the whole
OCx, using oc2-GAL4 (B), causes an expansion of the en-Z
domain (double-headed arrow, compare with A) and a reduction
in the size of the anterior Eya patch. In the adult, the en-Z signal
extends anteriorly (arrows, B′) around the reduced aOC
(compare with the control pattern in A′). In NP2631>OptixRI
discs (C) an anteriorward expansion of en-Z is also seen
(double-headed arrow). In adults of this genotype, the most
common phenotype is the unsplit aOC. In these heads, βgalactosidase signal is also seen more anteriorly, in between
the unsplit aOC (C′). The dashed lines (A′-C′) mark the
anteriormost limit of en-Z expression in controls. (D) OCx from
an en-Z; hh-GAL4/UAS-Optix late L3 larva, stained for Eya
(blue) and β-galactosidase (en-Z, red). Merged and individual
channels are shown. Arrow points to a region of weakened en-Z
expression where, in addition, Eya becomes derepressed.
the OCx, as observed. It is also important to mention that previous
work has shown that the Iroquois Complex (Iro-C) genes araucan
and caupolican participate in the restriction of the OCx to the medial
region (Yorimitsu et al., 2011) (Fig. 8).
Key to the establishment of OCx patterning is where en becomes
expressed. In contrast to hh transcription, which changes over time,
that of en is stable once initiated in the prospective IOC. We found that
Optix is expressed in a dorsal anterior strip in the eye disc, contained
within the otd domain (supplementary material Fig. S3), that abuts
posteriorly the en domain (Fig. 7). Our results suggest that Optix is
partially responsible for setting up this anterior border of en. Since en,
by acting as a Hh pathway repressor, prevents eya transcription, such
an expansion is the most likely cause of the effects on the aOC. The
definition of a clear-cut Optix/en border might be further refined by
mutually repressive interactions, as indicated by two results:
overexpressing Optix in the IOC results in the downregulation of en
(Fig. 7) and, reciprocally, the overexpression of en in the prospective
aOC represses Optix (supplementary material Fig. S4). Therefore, as
en is initially activated by hh, the anterior limit of the en domain may
result from the integration of activator and repressor inputs provided by
hh signaling and Optix, respectively, a limit that might be further
refined by reciprocal repression of Optix by en. We hypothesize that a
similar mechanism sets the posterior border of the en expression
domain to allow for an en-free, hh-receiving domain that becomes
specified as the pOC. Potential candidates for the posterior antirepressor are the Sp genes buttonhead (btd) and Sp1, and hunchback
(hb). These genes are expressed in the preoptic region of the embryonic
head of all arthropods (see Schaeper et al., 2010; Birkan et al., 2011;
Janssen et al., 2011; and references therein). However, neither Sp1 nor
btd is expressed in the prospective OCx region of the eye disc (Estella
et al., 2003; C. Estella, personal communication). We have checked for
hb expression using an anti-Hb antibody and found no expression in
L3 eye-antennal discs (not shown). Therefore, the nature of the
posterior regulator(s) involved remains to be determined. Fig. 8
summarizes these results into a schematic gene network (for an
extended network representation, see supplementary material Fig. S5).
Our results indicate that, despite their structural similarity and
shared requirement of otd, hh signaling and eya activation, the
patterning of the aOC and pOC are under different genetic control.
This might be expected, as only the anterior ocellar patches fuse
during metamorphosis to form the adult aOC. In fact, evidence for
this difference in genetic control had previously been obtained by
Maynard-Smith and Sondhi in population selection experiments in
Drosophila suboscura (Maynard-Smith and Sondhi, 1960). In this
study, the authors used an ocelliless (oc) mutant population that
showed loss of OCx structures, including the aOC and pOC, with
variable penetrance. Through breeding, they were able to establish
independent sublines in which the aOC, but not the pOC (or vice
versa), were preferentially lost, even when these flies were still
carrying the otd mutation. In light of these results, the authors
proposed that, on top of a common precursor for OC and bristle (i.e.
cuticle) determined by otd, an additional ‘system’ would control the
amount of ocellar or cuticle precursors, and this system would differ
along the anterior-posterior axis. In this context, Optix would not
instruct an ocellar fate but rather control the amount of anterior
ocellus precursor cells within the OCx primordium, thus acting as a
pre-patterning gene.
The expression pattern and function of six3/Optix have been
studied in Drosophila (Seimiya and Gehring, 2000; Coiffier et al.,
2008) and Tribolium (Posnien et al., 2009, 2011; Kittelmann et al.,
2013) embryos. In both insects, six3/Optix expression is restricted to
the head region, and includes the clypeolabrum and maxillary
Development (2015) 142, 2801-2809 doi:10.1242/dev.125179
Fig. 8. Gene regulatory network of OCx patterning. Schematics represent
the ocellar region of early (A), mid (B) and late (C) L3 discs. Medial (m) and
lateral (l) regions, and anterior (a) and posterior ( p) are indicated. The boxes
are color-coded and numbered to represent specific domains and depict the
regulatory relationships occurring at each stage. Genes are connected by
activating (arrows) or repressive (oval-ended) links. Open circles represent
the signaling pathway. Expressed genes/active links are colored/black,
whereas silent genes/inactive links are in gray. (A) Initially, broad wg
expression maintains otd in the anterior dorsal eye disc region. otd and wg
would then jointly activate hh transcription (1). The effects of wg signaling are
due, at least in part, to its downstream target, the transcription factor
homothorax (hth) (Brockmann et al., 2011), which has not been represented.
(B) hh expression builds up and triggers activity of the otd autoregulatory
enhancer oc7, causing an increase in Otd levels. High Otd then represses
wg. The result is a region devoid of wg, expressing high levels of otd and
uniform hh: the prospective ocellar complex region (OCx; 2). Uniform
expression of hh initiates the expression of one of its targets, eya, also
uniformly within the domain. In the absence of wg input, otd expression is
maintained through autoregulation, while maintenance of hh then relies on
another of its targets, en. These regulatory interactions only occur in the most
medial region of the disc. In more lateral regions, wg expression is
maintained (3); these will become the frons and periorbital cuticle in the adult
head. These mediolateral differences are regulated, at least in part, by the
Iroquois Complex (Iro-C) genes araucan and caupolican. High Otd drives the
expression of defective proventriculus (dve) to repress Iro-C genes which,
otherwise, would repress hh in the medial domain (Yorimitsu et al., 2011)
(2,3). The origin of this mediolateral asymmetry is not fully understood. (C) en
expression is restricted to a central domain. This confines hh production to
this central domain, the prospective IOC, as en is required to maintain hh
transcription (4). en becomes independent of hh signaling through a
mechanism that requires Dl/Notch signaling (Aguilar-Hidalgo et al., 2013).
Optix would restrict en expression anteriorly (aOC; 5). We propose the
existence of another repressor (‘?’) that represses en expression posteriorly
( pOC; 6). en represses the hh signal transduction cascade and this results in
the loss of eya from the IOC. In the aOC and pOC regions, eya expression is
maintained through an autoregulatory loop mediated by the Six1/2
transcription factor sine oculis (so) (which has not been represented for
simplicity) (5,6). However, it is also possible that Hh produced at the IOC acts
non-autonomously on the flanking aOC and pOC regions to activate eya
(dashed red arrows). The end result of this gene regulatory network is a
stable aOC-IOC-pOC domain organization in the disc.
2807
DEVELOPMENT
RESEARCH ARTICLE
RESEARCH ARTICLE
MATERIALS AND METHODS
Drosophila strains and genetic manipulations
The control strain used in this study was w1118. Reporter genes used were
hhP30-lacZ (Lee et al., 1992), wg-lacZ (Kassis et al., 1992), ey-lacZ (Xu et al.,
1999), oc7-lacZ (Blanco et al., 2009) and enXho25-lacZ (Hama et al., 1990),
all of which are described in FlyBase. The UAS/GAL4 system (Brand and
Perrimon, 1993) was used for most gain- and loss-of-function assays. We
used oc2-GAL4 (Blanco et al., 2009), 86C11-GAL4 (Jory et al., 2012), hhGAL4 (Tanimoto et al., 2000) and OptixNP2631-GAL4 (Hayashi et al., 2002)
lines, all of which showed expression in different domains of the dorsal head
during larval development. UAS lines used were: UAS-ArmS10 (UAS-Arm*)
(Pai et al., 1997), UAS-Hh6.1, UAS-OptixS1 (26806, Bloomington Stock
Center) and UAS-en (Guillen et al., 1995) for the ectopic expression
experiments; and UAS-enRNAi and UAS-OptixRNAi (35697 and 31910,
Bloomington Stock Center) for the knockdown inductions.
All crosses were raised at 25°C, except in the case of UAS-RNAi lines,
which were transferred to 29°C 48 h post-fertilization (hpf ) to maximize
the penetrance of the knockdowns. wg-GAL4 (Calleja et al., 1996) and
OptixNP2631-GAL4 were used as wg and Optix reporters to drive the
expression of UAS-GFP (Bessa and Casares, 2005) and UAS-lacZ
(Phelps and Brand, 1998), respectively. en loss-of-function clones were
generated through mitotic recombination (Xu and Rubin, 1993) in yw,
hs-flp; FRT42D Df(2R)enE/FRT42D, ubiGFP larvae. Df(2R)enE deletes
both the en and invected paralogous genes (described in FlyBase).
Clones were induced between 24 and 72 h after egg laying by a 45 min
heat shock at 37°C. Clones were marked in larval tissues by the absence
of GFP.
The hh lineage was followed using the G-TRACE method (Evans et al.,
2009). w; P(UAS-RedStinger)4, P(UAS-FLP.D)JD1, P(Ubi-p36E(FRT.
STOP)Stinger)9F6/CyO females were crossed to hh-GAL4 males and
raised at 25°C until dissection at 120 hpf.
The flip-out method (Struhl and Basler, 1993) was used to induce en gainof-function clones. Clones were induced 72-96 hpf by a 30 min heat shock
at 35.5°C in larvae from the cross of hsflp; act>y+>GAL4, UAS-GFP
females to UAS-en males.
2808
Immunostaining and imaging
Immunofluorescence in eye imaginal discs and embryos was carried
out according to standard protocols. Antibodies used were: guinea pig antiOtd (gift of Tiffany Cook, Cincinnati Children’s Hospital, USA; 1/1000),
rabbit anti-Optix (gift of Francesca Pignoni, Upstate Medical University,
Syracuse, USA; 1/500), guinea pig anti-Hb [gift of Karl Wotton (Kosman
et al., 1998); 1/2000], mouse anti-Eya (10H6; 1/1000), rat anti-CiA (2A1; 1/5)
[which detects the activator form of Ci (Aza-Blanc et al., 1997)] and mouse
anti-En (4D9; 1/15) from Developmental Studies Hybridoma Bank. lacZ
reporters were detected using a rabbit anti-β-galactosidase antibody (Cappel,
catalog number 55976; 1/1000). Appropriate Alexa Fluor-conjugated
secondary antibodies were used. Image acquisition was carried out using a
Leica SP2 AOBS confocal microscope. Images were processed with
Photoshop CS5 (Adobe).
Adult cuticle preparation
Dorsal head cuticle pieces were dissected from adult or late pharate heads in
PBS, and mounted in Hoyer’s solution:acetic acid (1:1) as described
(Casares and Mann, 2000). lacZ expression in enhancer trap line-derived
adult fly heads was monitored using the β-galactosidase substrate X-Gal as
described (Hama et al., 1990). Images were obtained using a Leica DM500B
microscope with a Leica DFC490 digital camera and processed with
Photoshop CS5.
Acknowledgements
We thank A. Iannini for technical assistance; E. Blanco and the Bloomington Stock
Center, Vienna Drosophila RNAi Center and Drosophila Genetic Resource Center
for fly lines; T. Cook, F. Pignoni and the Developmental Studies Hybridoma Bank,
University of Iowa, for antibodies; C. Estella (CBM-SO, CSIC-UAM, Madrid) for
communicating unpublished results; the CABD Advanced Light Microscopy Facility
for help with confocal imaging; and members of the F.C. laboratory for discussions.
Competing interests
The authors declare no competing or financial interests.
Author contributions
F.C. conceived the study and wrote the paper; M.A.D.-C. carried out most of the
research; M.A.D.-C. and F.C. designed the experiments and analyzed results.
Funding
This work was funded by the Spanish Ministry for Economy and Competitiveness
(MINECO) co-funded by European Fund for Economic and Regional Development
(FEDER) grants to F.C. [BFU2012-34324] and in which F.C. is a participant
[BFU2014-55738-REDT, BFU2014-57703-REDC].
Supplementary material
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.125179/-/DC1
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