RESEARCH ARTICLE 1301
Development 134, 1301-1310 (2007) doi:10.1242/dev.02807
The Zic family member, odd-paired, regulates the Drosophila
BMP, decapentaplegic, during adult head development
Heuijung Lee, Brian G. Stultz and Deborah A. Hursh*
The eye/antennal discs of Drosophila form most of the adult head capsule. We are analyzing the role of the BMP family member
decapentaplegic (dpp) in the process of head formation, as we have identified a class of cis-regulatory dpp mutations (dpps-hc) that
specifically disrupts expression in the lateral peripodial epithelium of eye/antennal discs and is required for ventral head formation.
Here we describe the recovery of mutations in odd-paired (opa), a zinc finger transcription factor related to the vertebrate Zic
family, as dominant enhancers of this dpp head mutation. A single loss-of-function opa allele in combination with a single copy of a
dpps-hc produces defects in the ventral adult head. Furthermore, postembryonic loss of opa expression alone causes head defects
identical to loss of dpps-hc/dpps-hc, and dpphc/+;opa/+ mutant combinations. opa is required for dpp expression in the lateral
peripodial epithelium, but not other areas of the eye/antennal disc. Thus a pathway that includes opa and dpp expression in the
peripodial epithelium is crucial to the formation of the ventral adult head. Zic proteins and members of the BMP pathway are
crucial for vertebrate head development, as mutations in them are associated with midline defects of the head. The interaction of
these genes in the morphogenesis of the fruitfly head suggests that the regulation of head formation may be conserved across
metazoans.
INTRODUCTION
The adult head of Drosophila is a complex sensory and feeding
structure. It is largely formed from paired eye/antennal imaginal
discs, which fuse with the clypeo-labral and labial discs during
metamorphosis to form a complete head (Bryant, 1978). The cells
that form each disc are set aside during embryogenesis and undergo
proliferation and pattern formation during larval life, before
differentiating during pupation.
The origin of the eye/antennal disc is complex, arising from
multiple embryonic segments (Jurgens and Hartenstein, 1993). Its
establishment of cell lineage restrictions differs from other discs,
with the dorsoventral boundary arising before that of the
anteroposterior (Baker, 1978; Morata and Lawrence, 1978; Morata
and Lawrence, 1979). Multiple functionally distinct structures, such
as the eye, antenna, maxillary palpus and head cuticle, arise from the
eye/antennal disc. The primordia for these structures appear to be
specified within the developing disc by localized patterns of
signaling molecules and regionally restricted expression of
transcription factors (Cavodeassi et al., 1999; Kenyon et al., 2003;
Pai et al., 1998; Pichaud and Casares, 2000; Royet and Finkelstein,
1996; Royet and Finkelstein, 1997).
Discs are comprised of two epithelial layers; a cuboidal disc
proper and an overlying squamous epithelium called the peripodial
membrane or peripodial epithelium. While the traditional view has
been that head structures arise primarily from the disc proper (J. L.
Haynie, PhD thesis, University of California, 1975) (Haynie and
Bryant, 1986), recent data suggest that the peripodial epithelium
plays a significant role in the development of the eye/antennal disc
(Cho et al., 2000; Gibson et al., 2002; Gibson and Schubiger, 2000).
At metamorphosis, the paired eye/antennal discs fuse into a single
Division of Cell and Gene Therapy, Center for Biologics Evaluation and Research,
Food and Drug Administration, Bethesda, MD 20892, USA.
*Author for correspondence (e-mail: [email protected])
Accepted 16 January 2007
vesicle and undergo complex morphogenetic movements inside the
pupal body cavity to form the head capsule, which everts to form the
final adult head (Fristrom and Fristrom, 1993; Milner and Haynie,
1979). How these morphogenetic movements come about and their
relationship to the underlying pattern elements is not understood.
We have undertaken a genetic analysis of adult head formation in
Drosophila. Our entrée into this was a specific class of
decapentaplegic (dpp) cis-regulatory mutations that affect only the
adult head capsule (Stultz et al., 2005). dpp is the Drosophila
homolog of Bone morphogenetic proteins (BMPs) 2 and 4, and the
major TGF␤-like protein in the fruitfly. The enhancer elements
disrupted in these mutations direct expression of Dpp in the lateral
peripodial epithelium of eye/antennal discs, and loss of this
expression in third instar eye/antennal discs results in defects in the
ventral head capsule (Stultz et al., 2006). We carried out an extensive
genetic screen to recover genes that interact with dpp to form the
ventral adult head (D.A.H., unpublished). Here we describe that one
interacting gene resulting from this screen is odd-paired (opa). opa
is a pair-rule gene (Jurgens et al., 1984; Nusslein-Volhard et al.,
1985) that encodes a zinc finger protein with homology to a family
of mammalian transcription factors, the ‘Zinc finger protein of the
Cerebellum’, or Zic family (Aruga et al., 1996; Benedyk et al., 1994;
Cimbora and Sakonju, 1995). Zic family members have roles in
neurogenesis, myogenesis, skeletal patterning and left-right axis
formation. In addition, a major role appears to be controlling regionspecific morphogenesis of the brain (reviewed by Aruga, 2004;
Grinberg and Millen, 2005).
In humans, mutations in Zic genes cause several congenital
cerebellar and head malformations, such as the Dandy-Walker
malformation (Grinberg et al., 2004) and holoprosencephaly (Brown
et al., 1998). In the fly, opa is required for the parasegmental
subdivision of the embryo, where it activates wingless and engrailed
in all parasegments (Benedyk et al., 1994). opa is also required for
the formation of all constrictions of the embryonic midgut, and it is
negatively regulated by dpp in this tissue (Cimbora and Sakonju,
1995). However, the postembryonic role of opa is completely
unknown.
DEVELOPMENT
KEY WORDS: decapentaplegic, BMP, odd-paired, Zic, Peripodial, Adult head
1302 RESEARCH ARTICLE
MATERIALS AND METHODS
Genetic strains and culture conditions
The following Drosophila melanogaster strains were used for this paper:
opa12.3, opa32.3, opaQ, opaM (this work), opa3D246 (Cimbora and Sakonju,
1995) (provided by Shigeru Sakonju), opa7 (opaIIC, provided by Steve
DiNardo, University of Pennsylvania Medical School, PA), opa8 (opa2P32,
provided by Trudi Schupbach, Princeton University, NJ), opats125
(Bloomington Stock Center), Df(3R)6-7, Df(3R)Z1, Df(3R)110 (provided
by Steve Wasserman, University of California, San Diego, CA), Df(3R)63
(provided by Steve DiNardo), dpps-hc1, dppTgR46.1, Df(2L)DTD2, P20
(Stultz et al., 2005), SH53 (dpps-hc-lacZ) (Stultz et al., 2006), ey-FLP
(Bloomington Stock Center), hsFLP;Sco/Cyo (provided by Mark Mortin,
NICHD/NIH, MD), FRT82B opa7 (this work), FRT82B Ubi-GFP
(Bloomington Stock Center), FRT82B Sb[63b] M(3) 95E Pr Bsb
(provided by Jim Kennison, NICHD/NIH, MD), UAS dpp (provided by
William Gelbart, Harvard University, MA), UAS opa (this work), MS1096Gal4 (Milan et al., 1998) (provided by Patrick Callaerts), c309-Gal4
(Bloomington Stock Center), and ey-Gal4 (provided by Francesca
Pignoni, Harvard Medical School, MA). Flies were maintained on standard
media and crosses were maintained at 25°C. Temperature shift
experiments were done using opats125, in combination with opa LOF
alleles. Crosses were temperature-shifted after first instar larval stage
from 18 to 29°C. Adult heads were fixed in 70% ethanol and mounted in
Euperol.
Scanning electron microscopy
Scanning electron microscopy was carried out using standard method as
described (Stultz et al., 2006).
Histochemical and immunohistochemical detection
␤-Galactosidase activity was detected by X-Gal staining as previously
described (Blackman et al., 1991). Discs were mounted in Aquamount
(Gurr), and examined using DIC. Adult heads were fixed in 1%
gluteraldehyde for 10 minutes, washed in PBT before staining. Heads were
incubated at 37°C in staining solution, plus 0.12% X-Gal dissolved in N,Ndimethyl formamide and 0.15% Triton X-100. Immunohistochemistry was
performed according to Carroll and Whyte (Carroll and Whyte, 1989).
Mouse anti-␤-galactosidase and anti-Dachshund (Developmental Studies
Hybridoma Bank) were used at 1:25 and rabbit anti-Odd-paired (provided
by Steve DiNardo) at 1:300. Secondary antibodies conjugated to Alexa Fluor
488 or Alexa Fluor 555 (Molecular Probes) were used at 1:1000. Nuclei
were visualized with DAPI. Discs were mounted in Vectashield (Vector
Laboratories) and imaged with a Radiance confocal microscope.
RNA in situ hybridization and fluorescence immunolocalization
RNA probes were labeled with digoxigenin (DIG) using the DIG RNA
Labeling Kit (Boehringer Mannheim). Antisense probe was transcribed by
T7 RNA polymerase from pKSII-opa (see Plasmid constructs), hydrolyzed
to an average length of 200-500 bp (Cox et al., 1984), precipitated in ethanol
and dissolved in hybridization solution. Imaginal discs were treated with
ethanol, xylene and acetone, as described (Nagaso et al., 2001). RNA was
detected with anti-DIG antibody (Roche Diagnostics), and signals were
detected using fluorescent secondary antibodies (Molecular Probes) and
imaged as above.
Plasmid constructs
opa cDNA was synthesized by PCR using an embryonic cDNA library
(Brown and Kafatos, 1988) as a template. PCR products were inserted into
pBluescript II KS(+) (Stratagene). This pKSII-opa was used for DIG-labeled
RNA probe. An EcoRI fragment of opa cDNA was cloned into the pUAST
vector (Brand and Perrimon, 1993). Transgenic flies were created by
standard protocols (Spradling, 1986).
Generation of genetic mosaics
Homozygous mutant opa somatic clones were generated by the FLP/FRT
technique (Xu and Rubin, 1993). An opa7 mutation was recombined with a
P[neo-FRT]82B chromosome to obtain P[neo-FRT]82B opa7. Recombinant
chromosomes were selected by resistance to G418 and lethality over opa
alleles. To produce opa clones in the imaginal discs, P[neo-FRT]82B opa7
was mated with eyFLP; P[neo-FRT]82B Ubi-GFP. Discs were dissected
and analyzed by confocal microscopy. opa clones were marked by loss of
GFP. To obtain opa clones in the adult head, P[neo-FRT]82B opa7 was
crossed to hsFLP; P[neo-FRT]82B Sb[63b] M(3) 95E Pr Bsb. Clones were
induced at 72, 96 or 120 hours after egg-laying by 1 hour heat shock at 38°C.
opa clones were identified by loss of markers for Stubble, Prickly and Blunt
short bristle.
RESULTS
opa participates with dpp in ventral head capsule
formation
We carried out a screen to recover mutations at dpp that specifically
affected the adult head capsule (Stultz et al., 2005). Several
mutations unlinked to dpp were recovered that behaved as dominant
enhancers of the dpp head capsule phenotype. Four of these
mutations were mapped to the third chromosome and, in addition to
causing a dominant interaction with dpp, also caused recessive
lethality. Cytologically visible breakpoints of three of these were
localized to region 82 on the polytene chromosomes, while the
fourth was mapped meiotically to a similar region (Table 1).
Deficiencies in this region were used to confirm that their recessive
lethality maps to this position and suggested that odd-paired was a
likely candidate. All four mutations fail to complement each other
and known opa alleles (Table 1). They all displayed embryonic
lethality and pair rule defects when crossed to each other and to
known opa alleles (data not shown).
The lethality and dominant interaction with dpp are caused by
mutations in a single locus. These four mutations, as well as
known opa loss-of-function (LOF) alleles and deficiencies that
remove opa, all interact dominantly with dpp head capsule
mutations to cause head defects (Table 1). In dpp head capsule
mutations, indicated as dpps-hc, the ventral head is disrupted, the
eye is round rather than oval, the sensory vibrissae on the ventral
side of the eye are bunched, the maxillary palps are missing,
reduced or duplicated, and gena tissue appears to be missing (Fig.
1B) (Stultz et al., 2005). This phenotype is also seen in mutant
DEVELOPMENT
Here we investigate the role of opa in adult head formation, and
its connection to dpp in this process. We find that opa is expressed
in the eye/antennal disc, primarily in the peripodial epithelium of
this structure. Loss of opa function during eye/antennal disc
development results in defects in ventral head structures identical to
those observed with loss of dpp. Expression of a peripodial-specific
dpp ␤-galactosidase reporter constructed from DNA from the cisregulatory region disrupted in dpp head capsule mutations is lost in
cells that do not express Opa, indicating that opa positively regulates
the peripodial dpp expression associated with ventral head
development. Targeted misexpression of Opa causes ectopic
expression of peripodial dpp, and dramatic head malformation.
These data indicate that dpp is regulated by opa, either directly or
indirectly, in a previously unknown pathway of head morphogenesis,
carried out in the peripodial epithelium, and suggests that the Zic
family role in head formation may be part of a conserved function
also seen in insects. Interestingly, holoprosencephaly caused by Zic
mutations in humans is autosomal dominant and has incomplete
penetrance. This behavior has been postulated to be caused by
digenic inheritance, with modifier genes enhancing the penetrance
of the Zic holoprosencephaly defect (Ming and Muenke, 2002). The
dominant genetic interaction we have observed between opa and
dpp, both of which have vertebrate homologs implicated in
holoprosencephaly, suggest that Drosophila head development may
be a model for this complex developmental genetic defect.
Development 134 (7)
opa in adult head formation
RESEARCH ARTICLE 1303
Table 1. Behavior of opa mutants
Mutant
12.3
opa
opa32.3
opaQ
opaM
opa deficiencies†,
loss-of-function*
Cytology
In(3LR)64D; 82E
Invisible, maps between scarlet
(73A1) and curled (86D3-4)
Df(3R)82B; 83A
In(3R)82B; 82E
opa allele*
dpps-hc1
Embryonic lethal
Embryonic lethal
Head capsule defects
Head capsule defects
Embryonic lethal
Embryonic lethal
Head capsule defects
Head capsule defects
Head capsule defects
combinations of opa LOF alleles with dpps-hc mutations (Fig. 1C).
In addition, when a temperature-sensitive opa allele, opats125, is
used to remove opa function postembryonically, similar defects
of the ventral head capsule are seen (Fig. 1D), along with
occasional antennal defects (see Fig. S1 in the supplementary
material). These data indicate that opa plays a postembryonic role
in the formation of the adult ventral head capsule, and that dpp
and opa function together in this process.
opa is expressed in the eye/antennal disc
We performed RNA in situ hybridization to the eye/antennal disc
using labeled opa as a probe. In the antennal disc proper, opa RNA
is expressed in a ring roughly coincident with the primordium of
the first antennal segment (Fig. 2A). It is also found on the lateral
side of the peripodial epithelium in the antennal disc, overlying the
primordia of the maxillary palpus and rostral membrane (J. L.
Haynie, PhD thesis, University of California, 1975) (Bryant, 1978;
Haynie and Bryant, 1986) (Fig. 2D). Peripodial expression extends
into the eye disc on the medial and lateral side. Additional
expression may also exist in the peripodial epithelium of the eye
disc, but it is not seen consistently. An enhancer trap from the 3⬘
end of the opa gene, opa3D246 (Cimbora and Sakonju, 1995), shows
similar ␤-galactosidase expression in the ring corresponding to the
presumptive antennal disc proper, and expression in the palps area
of the peripodial epithelium (Fig. 2B,C). We have made five large
␤-galactosidase reporter constructs from across the opa gene,
extending from approximately 5 kb upstream from the proteincoding region to approximately 6 kb past the end of the most 3⬘
coding exon (Fig. 3A). Three of these demonstrate significant
expression in the eye/antennal disc (Fig. 3B-D). This expression is
similar to that seen with our RNA localization and approximates
cumulatively to the expression of the opa3D246 enhancer trap, with
a ring of expression in the disc proper, and significant peripodial
expression along the lateral side of the disc. The expression of such
constructs is first detectable at the late second instar larval stage,
and increases subsequently (data not shown).
dpp expression responsible for the formation of the ventral head
capsule is limited to the lateral peripodial epithelium of the
eye/antennal disc (Stultz et al., 2006) (Fig. 4A; Fig. 5A); thus opa
expression in the eye/antennal disc is in the same epithelial layer as
dpp head capsule-related expression and on the same side of the disc
as the primordia of the future ventral adult head, although according
to the fate map these structures derive from the disc proper and not
the peripodial epithelium (J. L. Haynie, PhD thesis, University of
California, 1975) (Haynie and Bryant, 1986) (Fig. 2D). These data
place opa, a gene related to zinc-finger transcription factors, in the
correct location to be involved in the regulation of peripodialspecific dpp expression.
opa function is required for the correct expression
of dpp in the lateral peripodial epithelium
To assess the effect of opa on dpp transcription, we examined the
expression of a dpp ␤-galactosidase reporter construct, SH53, in the
presence of opa mutations. For simplicity, we will refer to this
reporter as dpps-hc-lacZ. The DNA in this reporter comes from the
dpp head capsule enhancer region, which resides in the 5⬘ cisregulatory region of the gene. It most accurately reflects the dpp
expression in the lateral peripodial epithelium of the eye/antennal
disc (Fig. 4A) that is responsible for the role of dpp in ventral head
formation (Stultz et al., 2006). In genotypes in which opa was
removed postembryonically, using opats125 at non-permissive
temperature, we observe a reduction in reporter expression,
indicating that opa is a positive regulator of dpp expression in this
location (Fig. 4B). This reduction was also observed in dpps-hc/+;
opa/+ mutant combinations (Fig. 4C,D), in agreement with the
dominant interaction between opa and dpps-hc described above (Fig.
1C; Table 1). Removal of peripodial dpp expression via homozygous
dpps-hc mutants produces an identical result (Stultz et al., 2006). dpp
has previously been observed to autoregulate its own expression in
several different tissues, including the eye/antennal disc proper
(Biehs et al., 1996; Chanut and Heberlein, 1997; Hursh et al., 1993;
Pignoni and Zipursky, 1997; Wiersdorff et al., 1996). These data
indicate that the transcription of dpp in the lateral peripodial
epithelium requires positive inputs from opa and the dpp signal
transduction pathway, and that the disruption in ventral head
formation observed in opa, dpps-hc or dpps-hc/+; opa/+ mutant
combinations all correlate to reduction of dpp expression in the
lateral peripodial epithelium. Expression was most strongly reduced
in a region near the lateral junction of the antennal and eye discs in
all these mutant combinations, midway along the band of dpp
peripodial expression. It is noteworthy that loss of opa function has
no effect on other dpp expression in the eye/antennal disc, as
monitored by the BS3.0 ␤-galactosidase reporter construct
(Blackman et al., 1991) (data not shown). The BS3.0 reporter
construct reflects dpp expression controlled by the 3⬘ cis-regulatory
region of the dpp gene. Mutants from this region of the dpp gene
(dppdisk alleles) do not interact with opa or dpps-hc alleles (Stultz et
al., 2005), indicating the regulatory autonomy of the lateral
peripodial expression domain. This indicates that opa positively
regulates dpp specifically in the lateral peripodial epithelium, and is
involved with dpp signal transduction related to ventral head capsule
formation, and not with other contributions of dpp to eye/antennal
disc morphogenesis.
As we were working with dpps-hc/+; opa/+ combinations, and
a temperature-sensitive opa allele, which might have residual opa
function, we wished to see if complete loss of opa function would
produce similar results on the dpp reporter construct.
DEVELOPMENT
Data represent the results of crosses of the mutant alleles listed in the left-hand column to the mutant alleles of the two right-hand columns.
*Two opa alleles were used: opa7 and opa8.
†
Deficiencies used: Df(3R)6-7, Df(3R)Z1, Df(3R)110 and Df(3R)63.
Fig. 1. opa and dpp interact genetically and produce identical
head capsule defects. Scanning electron micrographs of wild-type (A)
and head-capsule defect heads in opa and/or dpp mutants (B-G).
(B) dppTgR46.1/ Df(2L)DTD2, P20 (a strong dpp head capsule mutant
12.3
ts125
12.3
phenotype), (C) opa /+; dppTgR46.1/+ and (D) opa
/ opa .
12.3
(E) Missing palpus and disordered vibrissae from opa /+; dppTgR46.1/+.
(F) Enlargement of the palpus in C. (G) Enlargement of vibrissae region
ts125
12.3
in D. dpps-hc homozygotes, dpp/+;opa/+, and opa
/ opa
have
identical head capsule defects; eyes are smaller and rounder than
control (compare the length of brackets), vibrissae are disrupted and
clustered together (arrows), the maxillary palps are altered in shape and
in number (circles).
Homozygous opa7 clones, lacking endogenous opa activity, were
generated by using the FLP/FRT system (Xu and Rubin, 1993).
Somatic clones in the eye/antennal disc were produced in
eyFLP/+; P{neoFRT}82B P{Ubi-GFP}83 Ubi-GFP /
P{neoFRT}82B opa7 larvae and detected by loss of GFP. The
eyFLP construct has considerable expression in the peripodial
epithelium of the eye/antennal disc (Gibson et al., 2002). The
expression of dpps-hc-lacZ was lost in all opa7 LOF clones (Fig.
5B-D). There was complete correlation between LOF opa clones,
and loss of dpps-hc-lacZ expression, no matter what region within
the expression pattern the clone appeared, unlike our experiments
produced with the opa conditional allele, or with dpps-hc/+; opa/+
mutant combinations, where the midpoint of expression seemed
most sensitive to loss of dpp or opa function. Both large clones
(Fig. 5B,C) and small clones (Fig. 5D) displayed this correlation.
These data suggest that opa is required cell-autonomously for dpp
Development 134 (7)
Fig. 2. opa is spatially restricted in both peripodial epithelium
and cells of the disc proper in eye/antennal discs. The expression
of opa was examined by RNA in situ hybridization and
immunohistochemistry, and analyzed by confocal microscopy. The
antennal disc is up in both A and B. (A) opa RNA expression by
fluorescent in situ hybridization, 2D projection of confocal optical
sections through the eye/antennal disc. The bracket indicates nonimaginal disc cells (adepithelial cells or hemocytes) in the preparation
that also hybridize with opa probe. The arrow indicates medial disc
proper antennal ring; the arrowhead indicates lateral peripodial
epithelium staining. The lateral side of the ring in the disc proper is
obscured by the broad peripodial expression in this photo. (B) ␤galactosidase expression is directed by the opa enhancer trap opa3D246.
The white line shows the area of z-section, and corresponds to the xzimage shown in (C). The arrows indicate the antennal ring in the disc
proper. The arrowheads in B and C indicate peripodial epithelial
staining. (D) Fate map of third instar disc. ANT, antennal disc; EYE, eye
disc. (E) Schematic diagram of head structures. Shaded areas indicate
position of primordia on adult head. ANT, antennae; AR, aristae; PAL,
maxillary palps; VI, vibrissae.
expression in the lateral peripodial epithelium. We did, however,
recover larger clones at higher frequencies at the junction of the
eye/antennal disc. This is the same region that exhibited the most
pronounced decline in dpps-hc-lacZ expression in opa
temperature-shift experiments and dpps-hc/+; opa/+ mutant
combinations. Clones at the posterior end of the dpps-hc-lacZ
expression were recovered somewhat less frequently, and clones
in the most anterior region of dpps-hc-lacZ expression were
recovered rarely, and were quite small (Fig. 5E). We believe this
is due to the expression pattern within the peripodial epithelium
of the eyFLP construct. It is most heavily expressed in the ventral
peripodial epithelium at the connection between the eye and
antennal disc, overlapping dpps-hc-lacZ expression, but has more
limited expression in the peripodial epithelium of the ventral
antennal disc, and does not significantly overlap with dpps-hc-lacZ
in that region. These data extend our observation that opa is a
DEVELOPMENT
1304 RESEARCH ARTICLE
Fig. 3. Eye/antennal imaginal disc expression from opa Lac-Z
constructs. (A) Schematic diagram of the opa gene. Stippled boxes
represent exons and thin lines represent introns. Positions of five opa
constructs are indicated below the gene diagram. Position of the
opa3D246 enhancer trap is indicated by the triangle. (B-D) ␤galactosidase expression from (B) opa02 lacZ, (C) opa03 lacZ and (D)
opa04 lacZ, as detected by histochemistry. Lateral peripodial staining in
B-D is indicated by arrowheads, and staining of the disc proper ring in
D by an arrow. Note that B-D comprises the entire pattern seen in the
opa enhancer trap opa3D246 shown in Fig. 2B.
positive factor required for dpp expression in peripodial
epithelium of the eye/antennal disc, and further indicate that this
effect behaves in a cell autonomous manner.
We also wished to look at the effect of LOF opa clones on the
morphology of the adult head to see if the mutant phenotypes
observed with LOF clones resembled the data obtained with the opa
conditional allele, or with dpps-hc/+; opa/+ mutant combinations.
Somatic clones in adults were induced 72-120 hours after egg-laying
by 1 hour heat shock at 38°C of flies with the genotypes hsFLP/+;
P{neoFRT}82B P{piM} Sb[63b] M(3)95E Pr Bsb / P{neoFRT}82B
opa7. Defects in the adult head were observed only in clones
produced in the ventral portion of the adult head (Fig. 6A,B). LOF
opa clones in the dorsal head, identified by their bristle phenotype,
never displayed abnormal morphology (Fig. 6C). Results were
similar whether recombination was induced using eyFLP or hsFLP
constructs. Loss of ventral eye and rostral membrane tissue was
observed, as well as bunched vibrissae, and missing or misplaced
maxillary palps (Fig. 6A,B). Missing or malformed antennae were
often seen in adult clones, as had been seen in some opa conditional
allele experiments, but rarely in dpps-hc/+; opa/+ mutant
combinations and never with homozygous dpps-hc mutants. With the
exception of antennal defects, this spectrum of defects is similar,
although more extreme, to that observed with opats125 in temperature
shift-experiments and dpps-hc/+; opa/+ mutant combinations. LOF
opa clones more closely resemble defects observed in strong
dpps-hc mutants (Fig. 1B). Antennal defects, however, seem to be
specific to opa alone. In both temperature shifts and LOF opa
clones, multiple segments in the antennae are affected (Fig. 6A,B
and see Fig. S1 in the supplementary material), although the defects
are more severe in LOF opa clones, which often manifest complete
loss of antennal structures. We attribute antennal defects to the
expression of opa in the antennal disc proper, although the defects
RESEARCH ARTICLE 1305
Fig. 4. dpps-hc-lacZ expression is reduced in LOF opa mutants.
(A) Wild-type expression of the dpps-hc-lacZ reporter construct, SH53, is
seen on the lateral side of eye/antennal discs. Expression from this
reporter construct is reduced, most notably in the middle region (arrow)
ts125
12.3
12.3
in (B) opa
/ opa , (C) dppTgR46.1/+; opa /+ and (D) Df(2L)DTD2,
ts125
P20/+; opa
/+ (at 25°C) mutants.
extend further than the fate map would predict for the discreet ring
of opa expression. However, the function of opa in antennal
development does not appear to be part of the opa/dpp pathway
involved in ventral head development. These data further support the
postembryonic role of opa in forming the ventral adult head, and the
interaction of opa and dpp in ventral head formation.
Ectopic expression of opa results in ectopic
expression of dpp in the peripodial epithelium
We wished to see if ectopic expression of Opa was capable of
inducing dpp expression as monitored by the dpps-hc-lacZ reporter.
A full-length opa cDNA in a UAS expression construct was
ectopically expressed in imaginal discs, using the Gal4 expression
constructs MS1096 and c309. The MS1096-Gal4 driver expresses in
the peripodial epithelium and margin cells of the lateral and medial
sides of the eye/antennal disc (Fig. 7B) (Bessa and Casares, 2005).
Its ventral expression overlaps with that of the dpps-hc-lacZ reporter
(compare Fig. 7A with B). Ectopically expressing Opa with the
MS1096-Gal4 driver results in modest ectopic expression of the
dpps-hc-lacZ reporter in the peripodial epithelium on the medial side
of the disc (Fig. 7C). We compared the areas of ectopic dpps-hc-lacZ
reporter expression with the presence of Opa, using an Opa antibody
that is capable of detecting only overexpressed Opa. Areas where
ectopic reporter expression is detected by cytoplasmic expression of
␤-galactosidase were associated with the presence of nuclear Opa
protein (Fig. 7C,D,E). Nuclear Opa expression was also seen in
areas with no ectopic ␤-galactosidase expression (white arrows, Fig.
7C,D). These data suggest that Opa can induce dpp reporter
expression in the peripodial epithelium in a cell-autonomous
fashion, but that it either requires other factors that are not present in
all cells, or that the presence of repressors prevents Opa activation
in some areas. The c309-Gal4 driver expresses primarily in the disc
DEVELOPMENT
opa in adult head formation
1306 RESEARCH ARTICLE
Development 134 (7)
Fig. 5. opa LOF clones fail to express dpps-hc-lacZ. Homozygous
opa mutant clones were generated by using the FLP/FRT system.
dpps-hc-lacZ construct, SH53, expression (in red) in non-recombination
control disc (A), and in opa mutant tissue in single section images (B-E).
The clonal areas are outlined in white in the merged panels (B-D) and
are marked by absence of green fluorescence, which can be seen in the
B⬘-E⬘ green-only channel. The box in E indicates dpp reporter expression
missing in a small clone, and its enlargement is shown in the white
inset box in E and E⬘. The positions of clones relative to the entire
lateral dpps-hc-lacZ expression pattern is shown by labeled brackets in A.
proper, in the antennal portion of the disc, and behind the
morphogenetic furrow (Fig. 7G). Modest ectopic expression of the
dpps-hc-lacZ reporter is also seen with this driver. This expression is
limited to the peripodial epithelium, and is associated with a small
region of peripodial-specific expression of Opa, as identified by Opa
antibody (Fig. 7I,J). Note that in both cross sections (Fig. 7F,J), Opapositive nuclei in the disc proper are not associated with ␤galactosidase expression. Taken together, the results from both
drivers suggest that Opa has some ability to induce the expression of
the dpps-hc-lacZ reporter ectopically, but this ability is limited to the
peripodial epithelium. Additionally, not all areas that are Opapositive within the peripoidal epithelium are capable of expressing
dpps-hc-lacZ, while Opa expressed in the disc proper does not seem
to induce dpps-hc-lacZ expression in any area. We conclude that Opa
is not sufficient to induce reporter expression in all cells that express
the protein, either through lack of secondary factor(s), or due to
active repression in most areas of the disc. However, these results are
consistent with data obtained by the LOF clones, suggesting that opa
is a cell-autonomous activator of peripoidal dpp expression in certain
regions of the peripodial epithelium of the eye/antennal disc.
Adult heads of flies in which Opa was ectopically expressed using
either the MS1096 or c309 drivers were morphologically normal
(data not shown). We also used an ey-Gal4 driver to ectopically
express Opa to see what effect this had on dpps-hc-lacZ. The eyGal4>Opa eye/antennal disc is dramatically altered in shape, making
it hard to assess the effect of Opa ectopic expression on the reporter.
The antennal disc is duplicated, as indicated by markers of antennal
structures such as dachshund (Fig. 8F), and cut (data not shown),
and the eye disc is eliminated, with only a small amount of tissue
remaining (Fig. 8F,G). dpps-hc-lacZ also appears bifurcated in the
antennal region, but this may to be due to the fate change caused by
Opa ectopic expression rather than by induction of additional
dpps-hc-lacZ expression (Fig. 8G). The gross malformations in disc
DEVELOPMENT
Fig. 6. opa LOF clones display severe head malformations.
(A,B) Heat-shock induced clones in adult heads have small and round
eyes, abnormal antennae (arrowhead), vibrissae defects (arrow) and
missing palps (circle). (C) Clonal area in dorsal head, indicated by fulllength bristles, is normal.
opa in adult head formation
RESEARCH ARTICLE 1307
Fig. 7. Targeted misexpression of opa causes
ectopic dpps-hc-lacZ expression. (A) dpps-hclacZ expression (green) in a wild-type disc. (B) ␤galactosidase expression (red) driven by the
MS1096-Gal4. (C) Ectopic expression of dpps-hclacZ from MS1096-Gal4>Opa. Note boxed areas
of ectopic expression from the dpps-hc-lacZ
reporter on the medial side of the disc. The
white arrow indicates the region of Opa
expression where dpps-hc-lacZ is not expressed.
Cytoplasmic ␤-galactosidase expression (green)
and Opa overexpression (nuclear) as detected by
Opa antibody (red). (D,E) Higher magnification
images of the areas boxed in C. (F) Cross section
of area in E, showing that ␤-galactosidase
expression (green) and Opa antibody (red) are
colocalized within the peripodial epithelium
(arrow). (G) ␤-galactosidase expression (red)
driven by the c309-Gal4 driver. (H) Ectopic
expression of dpps-hc-lacZ from c309-Gal4>Opa.
A small area of ectopic ␤-gal expression is
boxed. (I) Higher magnification of the box in H.
Nuclear Opa (red) and cytoplasmic ␤galactosidase expression (green) are colocalized.
(J) Cross-section analysis of I. dpps-hc-lacZ and
Opa expression are again limited to the
peripodial epithelium (arrow). The nuclei of all
discs are stained with DAPI (blue). The peripodial
epithelium is oriented up in F and J, and lateral is
to the left in all pictures.
The expression of the ey-Gal4 driver is limited to the eye/antennal
disc, but we have observed that Opa causes morphological defects
in all discs when misexpressed with Gal4 drivers that express in all
discs. Opa appears to be a protein with potent morphological
abilities.
Cells from the ventral peripodial epithelium
persist in the adult, contributing to the ventral
head capsule
The primordia of the majority of the adult head structures are
reported to arise from the disc proper (Haynie and Bryant, 1986).
This would imply that the effects seen by disruption of peripodial
opa or dpp expression are caused solely by disruption of the
signaling required to support morphogenesis of structures derived
from the disc proper. To ask if the effect of opa and dpp on the adult
head was a secondary consequence of disrupted signaling to the disc
proper, or whether cells in the peripodial layer contributed directly
to structures in which we saw defects in opa and dpps-hc mutants, we
examined pharate adult heads for expression from the dpps-hc-lacZ
and our opa-lacZ reporter constructs. As monitored by
histochemical detection of ␤-galactosidase activity, cells from the
peripodial epithelium must persist in the adult head cuticle. In
dpps-hc-lacZ, the expression of which is limited to the peripodial
epithelium, significant ␤-galactosidase activity is seen in the ventral
head, including the anterior rostral membrane, maxillary palps and
a distinct area ventral to the prefrons, which abuts the clypeus (Fig.
9B). Light expression in the third antennal segment, and base of the
second antennal segment is seen with this construct. We cannot
currently explain this expression, as we do not see antennal defects
in dpps-hc mutations alone. The primordia of the Proximal rostral
sensilla (Prst) have been placed by the fate map in the lateral
peripodial epithelium (Haynie and Bryant, 1986) within the domain
DEVELOPMENT
structure are reflected by defects in the adult head. ey-Gal4>Opa
eliminates the eye. In the antennae, there are obvious duplications
of aristae, and antennal segments (Fig. 8B). Maxillary palps are also
duplicated (Fig. 8B,C). To determine if these dramatic head
malformations were primarily attributable to the induction of ectopic
Dpp expression in the peripodial epithelium by Opa, we expressed
Dpp using the same driver. ey-Gal4>Dpp also causes a dramatic
alteration in the antennal disc. dpps-hc-lacZ expression is broader,
extending medially, and dachshund expression indicates partial
antennal duplication (Fig. 8H,I). However, unlike ey-Gal4>Opa,
ectopic expression of Dpp on this driver does not eliminate the eye
disc, although it is reduced in size, and the retinal field is enlarged at
the expense of the rest of the disc. These disc alterations are reflected
in the defects seen in adult heads. The head is significantly reduced
in size, with misplaced and duplicated antennae and maxillary palps
(Fig. 8D). However, the eyes, while reduced, are not eliminated, and
protrude from the head. The dorsal head, although also reduced in
size, retains much of its normal appearance. The ey-Gal4 driver has
a band of expression in the peripodial epithelium extending from the
extreme anterior portion of the antennal disc into the eye disc (see
Fig. S2A in the supplementary material). However, it also has
extensive eye disc proper expression, particularly behind the
morphogenetic furrow, in the developing retina (see Fig. S2B in the
supplementary material). The difference between ey-Gal4>Opa and
ey-Gal4>Dpp may be attributable to different effects of these two
proteins in the eye disc proper. Opa appears to eliminate the eye disc
when expressed in the disc proper, while Dpp promotes retina
formation, a well-described function of dpp in eye development
(Dominguez and Casares, 2005). However, the effects observed for
both Opa and Dpp on the antennal disc are similar, thus may be
attributed to ectopic expression in the peripodial epithelium,
suggesting that the major target of Opa in this tissue layer is Dpp.
1308 RESEARCH ARTICLE
Development 134 (7)
Fig. 8. Opa misexpression causes severe head malformations.
(A) Wild-type head. (B,C) Adult heads from ey-Gal4>Opa. Eyes are
absent. Arrowheads indicate antennal and aristal duplications and
arrows indicate ectopic maxillary palps. (D) Adult head from eyGal4>Dpp. Eyes are present, but reduced. Arrowhead indicates
misplaced antenna, arrows indicate maxillary palps. Note duplicated
palpus on right. Asterisks represent outgrowths. (E) Wild-type and (F)
ey-Gal4>Opa third instar imaginal discs stained with antibody to
Dachshund. Note duplicated antennal ring, and lack of eye field
staining in the ey-Gal4>Opa disc. (G) dpps-hc-lacZ expression in eyGal4>Opa discs. Note the small amount of the remaining eye disc
(asterisk), and bifurcated dpps-hc-lacZ expression. (H) dpps-hc-lacZ
expression in ey-Gal4>Dpp discs. Staining no longer extends into the
eye disc, and extends further medially in the posterior antennal disc.
Compare with Fig. 4A. (I) ey-Gal4>Dpp discs stained with antibody to
Dachshund. Note partial duplication of antennal ring, and the
expansion of the retinal field anteriorly throughout the entire eye disc.
of dpps-hc-lacZ reporter expression. ␤-galactosidase expression is
seen in this region in posterior adult heads bearing the dpps-hc-lacZ
construct (Fig. 9C). The expression of the opa02 and opa03 lacZ
reporter constructs is also limited to the peripodial epithelium (Fig.
3B,C), and both show ventral head expression similar to the dpps-hclacZ reporter, with punctate expression in the maxillary palps (Fig.
9D,E). These two constructs have almost no expression in the
antennae. The opa04 lacZ construct, which has more extensive
lateral expression in third instar discs (Fig. 3D), has significant ␤galactosidase expression in the ventral head and maxillary palps
DISCUSSION
We recovered mutations in the gene opa as dominant enhancers of a
dpp mutant phenotype affecting ventral head development. In this
work we have established that this interaction arises due to the
requirement of opa for the expression of dpp in the peripodial
epithelium of the eye/antennal disc. In doing this, we have identified
a role for the pair-rule gene, opa, in the development of the
eye/antennal disc and subsequent ventral head morphogenesis.
Our work demonstrates that opa is an upstream activator of dpp
in the peripodial epithelium, and acts in a cell-autonomous fashion.
We do not know whether this role is direct, with Opa acting as a
transcription factor for dpp, or through other proteins. This ability to
activate dpp appears limited to the peripodial epithelium of the
eye/antennal disc, as misexpression of Opa in the disc proper does
not induce expression. Furthermore, Opa acts only on a dpp reporter
that has expression restricted to the peripodial epithelium of the
eye/antennal disc. With the exception of antennal defects, loss-offunction clones of opa produce identical head defects to
homozygous dpps-hc mutants, and ectopic expression of either Dpp
or Opa in the peripodial epithelium produces a similar spectrum of
misplaced sensory structures. These data suggest that dpp is the
major target of opa in the peripodial epithelium.
Both opa and dpp are involved in embryonic midgut
development, where dpp is a negative regulator of opa in the visceral
mesoderm (Cimbora and Sakonju, 1995). In addition, BMP2 and
BMP4 are negative regulators of Zic proteins in zebrafish (Grinblat
et al., 1998; Rohr et al., 1999), but the exact mechanism of this
regulation is unclear. Thus, Zic family proteins are often seen in
DEVELOPMENT
(Fig. 9F). This construct also expresses in the antennal portion of the
disc proper in the third instar, and has dark expression in the adult
third antennal segment, as well as light expression in the distal
portion of the second antennal segment. Heads from yw flies without
reporter constructs did not show ␤-galactosidase expression,
indicating that there is limited endogenous ␤-galactosidase activity
in the adult head, and an engrailed enhancer trap showed a pattern
of expression similar to that previously described (data not shown)
(Hama et al., 1990), which is distinct from the patterns generated by
our dpp and opa constructs. Thus we believe the expression we
observe accurately reports the contribution of peripodial cells to the
adult cuticle.
Fig. 9. ␤-galactosidase expression directed by peripodial reporter
constructs persists in adult heads. (A) Diagram showing front and
back of adult head with relevant structures labeled. Modified from
Bryant (Bryant, 1978). Prst indicates Proximal rostral sensilla.
(B-F) Histochemical detection of ␤-galactosidase activity in B, dpps-hclacZ, anterior view. (C) Same construct, posterior view. Arrows
represent Prst. Expression of (D) opa02 lacZ, (E) opa03 lacZ and (F)
opa04 lacZ.
regulatory networks with BMP proteins, but there does not seem to
be a canonical regulatory relationship. Our data indicates that during
eye/antennal disc development opa exerts a positive effect on
peripodial dpp.
Both opa and dpp exert their role on ventral head development
through expression limited to the peripodial epithelium of the
eye/antennal disc. The structures affected in ventral head capsule
mutations, such as palps and vibrissae, are reported to arise from
the disc proper in the fate map of the eye/antennal disc (Haynie
and Bryant, 1986); thus the effect of Opa-Dpp signal transduction
could be to cross epithelial layers, from the peripodial epithelium
RESEARCH ARTICLE 1309
to the disc proper. We have also shown that loss of lateral
peripodial Dpp expression results in apoptosis in the underlying
disc proper (Stultz et al., 2006), which further suggests a role for
peripodial signaling to support disc proper cell viability and
morphogenesis. However, when the descendants of peripodial
cells are followed by the perdurance of ␤-galactosidase
expression through metamorphosis, significant contributions of
lateral peripodial cells are found in areas of the ventral head where
we observe defects in dpps-hc or opa mutations, suggesting that the
ventral adult head is formed from descendants of both disc proper
and peripodial cells. Adult head expression has also been seen
with the MS1096-Gal4 driver, of which expression in the eye disc
is limited to the lateral and medial peripodial epithelium and
margin cells (Bessa and Casares, 2005). These data provide
further support to the idea that the peripodial epithelium provides
more than passive or purely mechanical functions during disc
development. The role of the peripodial epithelium in imaginal
disc development has begun to receive more attention, and there
is evidence that peripodial-specific signaling affects the patterning
of the eye (Cho et al., 2000), growth control (Gibson et al., 2002;
Gibson and Schubiger, 2000) and the fusion of discs at
metamorphosis (Agnes et al., 1999; Zeitlinger and Bohmann,
1999). It now seems likely that in addition to providing such
support to cells of the disc proper, peripodial cells contribute
directly to the cuticle of the adult head.
In mice and humans, Zic genes are associated with
holoprosencephaly, a congenital head defect the extreme
manifestation of which is cyclopia. In holoprosencephaly there is
variable loss or disruption in the development of the ventral
forebrain, and midline facial structures (reviewed by Muenke and
Beachy, 2000; Wallis and Muenke, 1999). Holoprosencephaly is a
common defect in humans, and genes in the TGF-␤ and hedgehog
pathways are also associated with both the human and mouse
condition (Hayhurst and McConnell, 2003; Petryk et al., 2004;
Zakin and De Robertis, 2004). Relevant to our work, a significant
number of holoprosencephaly cases result from autosomal dominant
inheritance, and often, obligate carriers of these autosomal dominant
pedigrees are clinically normal (Ming and Muenke, 2002; Nanni et
al., 1999). This incomplete penetrance suggests extreme dose
sensitivity and the presence of multiple modifying loci. The ability
of our genetic screen to recover multiple dominant enhancers of the
dpp ventral head defect, including opa, suggests that this may be a
model for the kind of digenic inheritance seen with
holoprosencephaly. The hedgehog pathway is known to be crucial
to adult head development in Drosophila (Royet and Finkelstein,
1996; Royet and Finkelstein, 1997), and our work adds TGF-␤ and
opa to this process in the fruitfly. It will be of interest to see how
many other connections exist between vertebrate and fly head
malformations.
We thank Steve DiNardo for sharing information and reagents during this
study. We thank Steve Wasserman, Trudi Schupbach, Shigeru Sakonju, Patrick
Callaerts, Francesca Pignoni, Jim Kennison, Mark Mortin and the Bloomington
Stock Center for fly stocks, and the Developmental Studies Hybridoma Bank
for antibodies. We are very grateful to Fernando Casares for sharing his
protocol for staining adult heads. We thank Tom Talbot for expert scanning
electron microscopy. We thank Judy Kassis, Mark Mortin, Mary Lilly and Brent
McCright for insightful comments on the manuscript. This work was funded
by the Center for Biologics Research and Review. This paper does not present
an official position of the FDA.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/7/2807/DC1
DEVELOPMENT
opa in adult head formation
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