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First published online 28 February 2007
doi: 10.1242/dev.02807
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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: deborah.hursh{at}fda.hhs.gov)
Accepted 16 January 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: decapentaplegic, BMP, odd-paired, Zic, Peripodial, Adult head
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
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 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 TGFß-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 region-specific 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.
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 ß-galactosidase reporter constructed from DNA from the
cis-regulatory 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.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
(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), MS1096-Gal4
(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
ß-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,N-dimethyl formamide and 0.15% Triton X-100.
Immunohistochemistry was performed according to Carroll and Whyte
(Carroll and Whyte, 1989).
Mouse anti-ß-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.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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).
|
). This phenotype is also seen in mutant 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 ß-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 ß-galactosidase reporter
constructs from across the opa gene, extending from approximately 5
kb upstream from the protein-coding 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 peripodial-specific
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 ß-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' cis-regulatory 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 ß-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. 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 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 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.
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). 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 ß-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 ß-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 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), Opa-positive nuclei in the disc proper are not associated
with ß-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
Opa-positive 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.
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).
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.
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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 ß-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 ß-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 of dpps-hc-lacZ reporter expression.
ß-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-hc- lacZ 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 ß-galactosidase expression in the
ventral head and maxillary palps (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 ß-galactosidase expression, indicating that there
is limited endogenous ß-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.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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-of-function 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 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.
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); thus the effect of Opa-Dpp signal transduction
could be to cross epithelial layers, from the peripodial epithelium 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 ß-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-ß
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-ß 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.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/7/2807/DC1
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ACKNOWLEDGMENTS |
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