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First published online 28 May 2008
doi: 10.1242/dev.017053
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Centre de Biologie du Développement, UMR 5547 and IFR 109, Université de Toulouse and CNRS, 118 route de Narbonne, Bâtiment 4R3, 31062 Toulouse Cedex, France.
* Author for correspondence (e-mail: cribbs{at}cict.fr)
Accepted 7 May 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Wingless, Eye-antenna disc, Spineless, Temporal regulation, Maxillary palps
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
eye-antennal (E-A) imaginal disc gives rise to the major head sensory organs
(eyes, ocelli, antennae and maxillary palps)
(Haynie and Bryant, 1986),
making it a model for the development of multiple organs from a composite
rudiment. From the second larval instar onwards, the complementary expression
domains of the transcription factors eyeless (ey)/twin
of eyeless (toy) and cut (ct) define fields
that ultimately yield the eye and antenna, respectively
(Dominguez and Casares, 2005;
Kenyon et al., 2003). Axial
organisation of the antenna employs a canonical genetic cascade typical of
ventral appendages, analogous to the one described for legs
(Brook et al., 1996). This
cascade starts with the expression of the homeobox selector gene
engrailed in the posterior compartment, where it specifies posterior
cell fate and activates transcription of the hedgehog (hh)
gene. Hedgehog protein secreted by posterior cells diffuses across the AP
boundary to nearby anterior cells, where it activates the transcription of
target genes, including decapentaplegic (dpp/TGF-β)
anterodorsally and wingless (wg/Wnt) anteroventrally
(Diaz-Benjumea et al., 1994;
Lecuit and Cohen, 1997).
Mutual antagonism between these two morphogen growth factors is necessary to
activate expression of the homeodomain transcription factor Distal-less (Dll)
in the centre of the disc, thereby setting the proximodistal (PD) axis
(Diaz-Benjumea et al., 1994;
Lecuit and Cohen, 1997).
Activation of Dll in the antennal disc leads to the co-expression of
Dll and the TALE homeodomain protein Homothorax (hth; the
Drosophila Meis1 homolog), whereas they do not overlap in the leg
disc (Dong et al., 2001). Two
lines of argument indicate that Dll and hth jointly specify
antennal fate. First, haploinsufficient alleles of Dll or mitotic
clones of hth- cells can transform antennae into legs
(Casares and Mann, 1998;
Dong et al., 2000;
Dong et al., 2001). Second,
ectopic co-expression of Dll and hth can cause
transformations to antenna (Dong et al.,
2000; Dong et al.,
2002). Dll and hth are thus required,
respectively, for the specification of the distal versus the proximal domains
of ventral discs but also for the specification of antennal fate
(Abu-Shaar and Mann, 1998;
Dong et al., 2000;
Dong et al., 2002;
Wu and Cohen, 1999). They
regulate multiple target genes during antennal development that function in
specifying antennal structures and/or repressing leg development
(Casares and Mann, 1998;
Dong et al., 2000;
Dong et al., 2002). One of
these targets is spineless (ss), which encodes a b-HLH-PAS
protein homologous to the mammalian Aryl Hydrocarbon Receptor
(Duncan et al., 1998).
ss is considered to be an antennal selector gene as the antennae of
ss- adults are transformed to distal legs while the
mis-expression of Ss induces ectopic antennal structures in adult legs
(Dong et al., 2002;
Duncan et al., 1998;
Struhl, 1982). Ss
controls distal antennal differentiation at least in part via the activation
of its target genes distal antenna (dan) and distal
antenna related (dan-r) that encode nuclear `pipsqueak' motif
proteins (Emerald et al.,
2003). Ectopic expression of dan or dan-r causes
a partial transformation of distal leg towards antenna, whereas loss of both
functions has the opposite effect (Emerald
et al., 2003; Suzanne et al.,
2003). They are thus considered to be effector genes of antennal
identity.
Though the antennal imaginal disc has often been treated as a single
cellular field giving rise to the adult antenna, it has long been known that
the second olfactory organ of the adult head - the maxillary palp - also
originates from the antennal disc (Haynie
and Bryant, 1986). A maxillary (Mx) primordium that gives rise to
the adult maxillary palp emerges from the antennal disc as a localised
outgrowth in early pupae (Jurgens and
Hartenstein, 1993). However, very little has been described
concerning the developmental origins of the Mx palp, nor of the genetic
program leading to its final form. Two of the antennal determinant genes,
ss and Dll, are known to be required for Mx development, as
loss of their functions leads to adults with reduced or deleted Mx structures,
respectively (Cohen and Jurgens,
1989; Duncan et al.,
1998). Consistent with these mutant phenotypes, both Dll
and ss are expressed at the pupal stage in the Mx primordium of the
antennal disc (Duncan et al.,
1998; Panganiban,
2000). Contrary to the antenna, recent work places Dll
downstream of ss in the maxillary field
(Emmons et al., 2007).
Additionally, the homeotic genes proboscipedia (pb;
HoxA2/B2) and Deformed (Dfd; Hox A4-D4) are both expressed
in the Mx primordium (Benassayag et al.,
2003; Diederich et al.,
1991) and required there, as adult Mx palps are deleted by mitotic
Dfd- clones, and reduced in pb-
homozygotes (Merrill et al.,
1987; Pultz et al.,
1988). As ectopic pb expression transforms distal
antennae into Mx palps, pb is considered to be a Mx selector gene
(Cribbs et al., 1995). The
reciprocal transformation of Mx into antennae can be observed when ss
or wg are mis-expressed in Mx cells
(Duncan et al., 1998;
Johnston and Schubiger, 1996).
Thus, the antennal and Mx organs appear to be homologous structures that
emerge from the same imaginal disc, share key selector genes involved in their
specification and both contribute to adult olfactory function.
In this paper, we address the issue of regional specification of the Mx field within the antennal disc. We find that the Mx field is defined by Deformed expression from the second larval instar onwards. The program for Mx regionalisation that emerges here is a temporally deferred version of the antennal program, owing to the delayed expression of the ventral signal Wg in the prepupal Mx field. We show that precocious wg expression in this tissue is sufficient to transform Mx to antenna, indicating that the delayed Wg expression in the Mx primordium is crucial in distinguishing antennal and Mx identity. Finally, our analysis reveals that Wg acts through ss in the maxillary field, but wg can also influence organ identity independently of the ss selector function.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Drosophila strains and transgenic lines
The strain used as wild type in this study was w. Reporter genes
used were dpp-lacZ BS3.0 (Blackman
et al., 1991) and ptc-lacZ
(Zhang and Kalderon, 2000).
The ss null allele ssD115.7
(ss-) is described by Duncan et al.
(Duncan et al., 1998). For
targeted mis-expression, ptc-GAL4 driver line
(Hinz et al., 1994) was
combined with responder constructs UAS-ss
(Duncan et al., 1998) or
UAS-wgts M7-2.1
(Wilder and Perrimon, 1995).
The fly strains employed for clonal analysis were: (1) w; FRT82B, (2)
hs-FLP, w; FRT82B Ub-GFP, M(3)RpS3/TM6B, Hu Tb, (3) y w
hs-FLP; FRT82B pygo130/TM6B, Hu Tb, (4)
y hs-FLP; FRT42D Ub-GFP/Cyo, and (5) y w; FRT42D
DllSA1/SM5-TM6B, Hu Tb. Stocks specifically constructed for
this work were: (1) ptc-GAL4; ss-/TM6B, Hu Tb, (2)
UAS-wgts, ss-/TM6B, Hu Tb, (3) hs-FLP;
ptc-GAL4; FRT82B pygo130/TM6B, Hu Tb, and (4) UAS-ss;
FRT82B Ub-GFP RpS3/TM6B, Hu Tb.
Conditions for temporal wgts activation
After crossing ptc-GAL4 and UAS-wgts flies,
egg lays were collected for 24 hours, and then adults were removed to fresh
medium. Development is slowed for this genotype
(Johnston and Schubiger,
1996). Developing animals were maintained at 25°C for 1 day
(
L1), 2 days (L2), 3 days (early L3), 4 days (mid L3), 5
days (late L3) or 6 days (early pupa), then shifted to 18°C to
activate wg. Dan staining was performed on imaginal discs from late
L3 larvae or from pupae, depending on the timing of wg activation.
For molecular analysis the wggof and wggof,
ss- larvae (ptc-GAL4/+; FRT82B
ss-/UAS-wgts, ss-) were shifted to
18°C during L3 stage, after 4 or 5 days of development.
Clonal analysis
Clones were generated using the FLP/FRT system
(Xu and Rubin, 1993).
For lineage analysis, Minute-enhanced mitotic clones were induced in hsFLP;FRT82B/FRT82BUb-GFP, M(3)RpS3 animals by a single 30-minute heat shock at 38°C (condition where there is one clone per antennal disc) after about 1 (L1), 2 (L2), or 3 (L3) days of development.
In the other experiments, clones were generated by a single 1-hour heat shock at 37°C during the first or second larval instar, then dissected and stained in late third instar or in pupal discs: in hs-Flp; FRT82B pygo130/FRT82B Ub-GFP, M(3)RpS3 animals for pygo mutant clones; in hs-Flp; ptc-GAL4/UAS-ss; FRT82B pygo130/FRT82B Ub-GFP, M(3)RpS3 (ssgof, pygo-) for pygo mutant clones overexpressing ss; or in hs-FLp; FRT42D DllSA1/FRT42D Ub-GFP for Dll mutant clones.
Immunocytochemistry and antibodies
Larvae or white pupae were prepared for immunofluorescence essentially as
described by Agnes et al. (Agnes et al.,
1999). All incubations were performed without agitation to avoid
damaging the pupal tissues. Primary antibodies used were: mouse anti-Cut 2B10
(1/200), concentrated anti-Inv 4D9 (1/20) and anti-Wg 4D4 (1/200) from the
Developmental Studies Hybridoma Bank (DSHB); mouse anti-Dll, 1/500 (I.
Duncan); rabbit polyclonal anti-Dfd, 1/250 (T. Kaufman); rabbit anti-Hth,
1/250 (N. Azpiazu); rat polyclonal 
-Dan, 1/300 (S. Cohen); guinea pig
anti-Ss, 1/1000 (L. Jan and Y. N. Jan); and rabbit anti-βGal, 1/5000
(Cappel, Promega). Mounted discs were viewed using a Zeiss LSM410 or a Leica
TCS SP2 confocal microscope.
Adult cuticle analysis
Flies of interest were stored in ethanol until dissection. Detailed
examinations of dissected heads mounted in Hoyer's medium were performed by
light microscopy on a Zeiss Axiophot.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Two Hox genes required for Mx differentiation,
proboscipedia (pb) and Deformed (Dfd)
(Merrill et al., 1987;
Pultz et al., 1988), are
expressed in the Mx territory: Pb is initiated in the pre-pupal Mx primordium
and Dfd is present in L3 (Benassayag et
al., 2003; Diederich et al.,
1991). We therefore examined expression of the Dfd protein as a
potential molecular marker to follow maxillary development within the antennal
disc. Dfd already accumulates in maxillary cells in early L2 larvae, the
period at which segregation of the eye and antennal territories occurs
(Kenyon et al., 2003), in a
pattern complementary to the antennal field marker Cut
(Fig. 1A). This pattern of
exclusion between Cut and Dfd then persists both in the disc proper and in the
peripodial membrane through L3 (Fig.
1B), in early pupae (Fig.
1C), and into metamorphosis, where Dfd remains in the Mx palps of
a pupal head after fusion of the eye-antennal and labial discs
(Fig. 1D, arrows). We conclude
that Dfd marks a Mx field that is present in the antennal disc from the L2
larval stage onwards into pupal development.
To test whether the exclusive patterns of Dfd and Cut markers reflect
separate cell populations, we examined growth-enhanced wild-type mitotic
clones that touch the Dfd/Cut limit (in the posterior antennal disc; see
Fig. 1B,C). In the conditions
used, most antennal discs contained one or no clones. Most clones induced from
L2 onwards were restricted to the maxillary (12/33;
Fig. 1E) or the posterior
antennal territory (12/33; Fig.
1F). Another class of clones circumnavigates the posterior antenna
to include maxillary and anterior antennal cells (6/33; not shown), consistent
with previous observations (Morata and
Lawrence, 1979). Only 3/33 L2-induced clones encompassed both
posterior antennal and maxillary cells, while a significantly larger
proportion of clones induced in L1 larvae did (18/59 clones). This difference
indicates that the expression domains detected with Dfd and Cut markers in
early L2 reflect the establishment of a clonal restriction between Mx and
antennal fields.
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Initial anteroposterior specification involves expression of engrailed/invected (en/inv) that determines posterior cell identity and activates transcription of the hedgehog (hh) morphogen there. In L2 larvae, the single group of cells expressing En/Inv/Hh proteins in the antennal disc is restricted to the antennal territory, as it does not overlap with Dfd-expressing cells (Fig. 2A,B; data not shown). Unexpectedly, the Hh transcriptional target patched (ptc) is activated on both sides of these posterior antennal cells, bordering them in adjacent anterior antennal cells but also in the maxillary field (Fig. 2F,G; as seen by a ptc-lacZ transgene that recapitulates normal ptc expression). The same result was obtained with antisera directed against Ptc protein or the activator form of the Hh target protein Cubitus interruptus (Ci) (not shown). These data strongly suggest that both antennal and maxillary cells receive and transduce diffusible Hh signal from a common antennal source at this stage. The first evidence for AP compartmentalisation of the maxillary field is detected in early L3 larvae, when En/Inv co-expression with Dfd is observed (Fig. 2C, arrowhead), and is clearly detectable in late L3 larvae and in pre-pupae (arrowhead in Fig. 2D,E). This zone of En-expressing cells largely excludes the pre-existing stripe of ptc-lacZ expression during L3 and pupal stages (arrowhead Fig. 2H-J).
Concerning DV axis organisation, dpp-lacZ and wg
appear simultaneously in the antennal territory in early L2
(Fig. 2K). This expression in
two adjacent wedges is maintained through L2, L3 and into pupal stages
(Fig. 2L-O). Organisation of
the PD axis is not yet noted in early L2 larvae, where Hth is uniformly
distributed across the antennal disc, while Dll is absent
(Fig. 2P). Immediately
following the onset of dpp and wg expression during L2,
Dll (Fig. 2Q) and
dachshund (dac; not shown) are activated, while Hth retracts
from the centre of the disc (Fig.
2P,Q) giving rise during L3 to distinct domains defined by Dll
alone, joint Dll/Hth and Hth alone (Fig.
2R-S) (Dong et al.,
2001).
In the maxillary field, ptc expression in late L2 (Fig. 2G) is not accompanied by dpp or wg (Fig. 2L). dpp-lacZ expression in the maxillary territory appears in early L3 larvae (Fig. 2M). By contrast, Wg is absent throughout L3 development (Fig. 2M,N), and only appears nearly 2 days later at the L3/pupal transition, in anterior maxillary cells, adjacent to (and exclusive of) those expressing Dpp (Fig. 2O). Mitotic hh- clones confirmed that this maxillary wg expression is hh dependent (not shown). In prepupae, Dll appears in a group of Mx cells centred on the Dpp-Wg junction that largely overlaps Hth there (inset, Fig. 2T). Dac is not detected in the Mx primordium (not shown). These data, summarised at the bottom of Fig. 2, indicate that the maxillary region deploys a program similar to the antennal program but delayed by the late appearance of Wg.
Timing of wg signalling defines maxillary versus antennal identity
Temporally regulated Wg thus might play a key role in distinguishing the
genetic programs leading to maxillary and antennal fates. One described
consequence of mis-expressing Wg is the transdetermination of maxillary palps
to antennae (Johnston and Schubiger,
1996). We re-examined this effect of Wg on Mx/Ant identity, paying
particular attention to the temporal activity of Wg. A conditionally active
Wgts protein that is secreted at 18°C but not at 25°C
(Gonzalez et al., 1991;
Wilder and Perrimon, 1995),
was driven by ptc-Gal4 for varied times and durations at 18°C
(Fig. 3; see also Materials and
methods). Mx-to-Ant transformations were scored by two criteria: (1)
expression of antennal markers, especially Dan, in the Mx territory of the E-A
disc (Fig. 3A), and (2)
appearance of identifiable adult antennal tissue, notably aristae, in place of
Mx palps (Fig. 3B, arrow).
ptc-Gal4>UAS-wgts animals that develop at 25°C eclose as normally patterned adults (not shown). Animals raised at 18°C from embryogenesis onwards all died before L2. By contrast, shifting from 25 to 18°C during larval development yielded a Mx-to-Ant transformation whose frequency was strongly influenced by timing (see table in Fig. 3). When the passage to 18°C was carried out in L1 larvae, we observed significant numbers of transformed larvae/pupae and adults (28% of Dan-expressing discs, 53% of adults with Mx-to-Ant transformation). The penetrance of the transformation was markedly enhanced when the permissive temperature was installed later, in L2 (63% and 85%), early L3 larvae (73% and 90%) or mid-L3 (83% and 89%), then maintained until adult eclosion. Thus, the most efficient conditions leading to Mx-to-Ant transformation were those where Wg was overexpressed concomitantly with Dpp in Mx cells (see Fig. 3).
Temperature shifts to 18°C of pre-pupae produced almost no transformation (6% and 7%). This result shows that overexpression of the Wg morphogen in pre-pupae is not sufficient to direct Mx-to-Ant transformation, and indicates that precocious Wg activation is a crucial initiator of Mx-to-Ant transformation. We infer that the rare individuals obtained with Mx-to-Ant transformation under this condition have probably been subjected to Wg overexpression during the late L3 stage, in light of the 24-hour egg-lay periods employed (see Materials and methods). Finally, when transgenic Wg was expressed solely during the L3 larval stage, no transformed individuals were obtained (0/26), suggesting that continuous expression of Wg and/or a precise spatial pattern of ectopic expression are required for efficient transformation. These results were confirmed using the flip-out technique to generate new sources of Wg that do not depend on the ptc promoter. Wg-expressing clones (act>y+>wg) induced by hsFlp in second instar larvae were sufficient to trigger aristal formation on adult Mx palps and ectopic Dan expression in the L3 larval Mx field (not shown). Taken together, these results indicate that temporal control of wg activity in pre-pupae versus larvae is crucial to distinguishing the Mx-specific developmental program from its antennal counterpart.
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). We
therefore compared the temporal and spatial maxillary expression of Wg and Ss.
In the wild-type Mx territory, both Wg and Ss are absent in L3
(Fig. 2M,N; not shown), then
appear simultaneously there at the L3-prepupal transition
(Fig. 4A), followed in prepupae
by Dll activation in a subset of ss-expressing cells
(Fig. 4B). Mis-expressed Wg
induces a precocious Mx expression of Ss and Dll
(Fig. 4C; not shown).
Conversely, mis-expressing Ss (ptc-Gal4>UAS-ss) resulted in
precocious Wg accumulation in L3 larvae that is first detected in the band of
ptcGal4>ss-expressing cells (not shown) and then resolved to an
antennal-like wedge pattern (Fig.
4D). This ss-dependent Wg activation starts in early L3
larvae, concomitant with and adjacent to endogenous dpp expression
(not shown), and leads to earlier activation of Dll protein in the Mx field
(not shown). These gain-of-function experiments strongly underline the
capacities of Wg and Ss for mutual activation. Furthermore, both Wg and Ss
direct a remarkably rapid reorganisation of the Mx territory towards an
antennal primordium during L3 (Fig.
4C,D), though Wg seems more potent in this respect.
We therefore examined the relationship between wg and ss
using loss-of-function mutations. Both affect maxillary development, as
ss- homozygotes and adults deficient for wg
signalling harbour reduced palps (Duncan
et al., 1998) (and not shown). In ss- mutant
pupae, Wg and Dll proteins accumulate at roughly normal levels in the Mx
primordium (Fig. 4E,F).
Conversely, when wg signalling was abolished via mitotic clones that
eliminate the obligatory pathway element pygopus
(Belenkaya et al., 2002), Ss
(Fig. 4G) and Dll (not shown)
were absent from mutant pygo- maxillary cells. The
cell-autonomous loss of ss expression in pygo-
maxillary cells indicates that wg signalling is required for
ss activation there. Although we have not directly tested the role of
dpp signalling, these loss-of-function results support a model where
dpp/wg signalling acts upstream of Dll and
ss in the maxillary palp, as for other ventral appendages.
Furthermore, in Mx Dll- mutant clones, Ss is expressed
normally (Fig. 4H). This
suggests that, contrary to the antenna, Mx ss expression is
independent of Dll.
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wg controls the maxillary-versus-antenna identity choice via ss-dependent and -independent activities
The complex interactions revealed by the preceding results led us to
examine the relationship of wg and ss in maxillary/antennal
development through functional tests of epistasis. In ss-
mutants, antennae are transformed to distal legs
(Duncan et al., 1998) and
maxillary palps are reduced relative to wild type (compare
Fig. 5A1,
5A2), whereas
ptc-directed Wgts (wggof) induces a
highly penetrant Mx-to-Ant transformation
(Fig. 3B). Most double-mutant
wggof; ss- adults harbour a large
nondescript maxillary outgrowth (Fig.
5A3), but in some the Mx was replaced by a tarsus
culminating in two distal claws (Fig.
5A4). That the maxillary field can be re-directed by Wg
to a leg in the ss- condition suggests that mis-expressed
Wg has reorganized the maxillary region into a permissive `pre-antennal'
environment whose transformation to antenna is blocked by the absence of
ss activity (see Fig.
5G). We therefore examined the molecular organisation of
wggof; ss- larval E-A discs, using Dfd
and Dan as Mx and antennal markers, respectively
(Fig. 5B). Singly,
wggof (Fig.
3A, Fig. 5C) and
ssgof (Fig.
5D) induced a similar reorganisation of the Mx region to antenna,
visible in L3 larvae by retraction of Dfd and de novo Dan and Dac accumulation
(Fig. 5C,D; not shown). By
contrast, in wggof; ss- larvae, Dan was not
observed in either the antennal or the maxillary field
(Fig. 5E), correlating with the
non-antennal adult Mx outgrowth (Fig.
5A3,4). This indicates that mis-expressed Wg requires
normal ss function to induce Dan and the subsequent antennal program.
However, Dfd was reliably retracted from the budding `maxillary' region of the
same wggof; ss- animals
(Fig. 5E, arrowhead), similar
to wggof alone (Fig.
5C). This retraction of Dfd indicates that Wg signalling can
reorganise the Mx field independently of Ss.
In the reciprocal test, pygo- clones were used to remove Wg signalling activity from ssgof tissues. No adults were obtained under these conditions, but Minute-enhanced pygo- clones could be obtained in the antennal-maxillary region of ssgof larvae (Fig. 5F,F'). As described above, pygo- clones lead to inactivation of ss (Fig. 4G) and its target gene dan (not shown). In ssgof; pygo- double mutant cells, Dan was activated only in the narrow band of cells expressing Ss under the ptc-Gal4 driver but not in the wider concentric rings typical of the Mx-to-antennal transformation (compare Fig. 5F with 5D), whereas normal Dfd expression persisted throughout the Mx field (Fig. 5F'). Ss protein is thus sufficient to induce Dan expression but requires wg signalling activity to achieve the Mx-to-Ant transformation, as seen by tissue morphology, molecular reorganisation of Dan-expressing regions and persistence of Dfd. Taken together, these results indicate that Wg provides a crucial input to the program initiated by Ss, but can also contribute to identity choice independently of Ss.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The antennal imaginal disc is divided early into distinct but communicating antennal and maxillary territories
Much previous attention for the eye-antennal disc has been directed towards
the separation into eye and antennal fields
(Dominguez and Casares, 2005;
Kenyon et al., 2003;
Kumar and Moses, 2001).
However, how the antennal region gives rise to both the antenna and the
maxillary palp has not been addressed. We found that expression of Dfd (Mx)
and Cut (Ant) define clonally separate antennal and maxillary fields of the
antennal disc established by L2, at roughly the same time as the eye-antennal
demarcation. Morata and Lawrence (Morata
and Lawrence, 1979) showed that adult clones induced as late as
third instar larvae can encompass the Mx palp and anterior antennal cells. The
clonal boundary we found in imaginal discs, separating the maxillary field
defined by Dfd expression from posterior antennal cells, is compatible with
their conclusion. The antenna is constructed through a program initiated by En
and relayed by Hh. Maxillary and antennal cells appear to share this Hh source
during L2, as (1) En/Hh is limited to antennal cells at this stage
(Fig. 2A,B), (2) this antennal
Hh source informs cells on both sides, as seen by ptc expression
(Fig. 2F,G), and (3) mitotic
hh- clones show that Mx ptc and wg
expression is dependent on antennal hh signalling (not shown).
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) that ss contributes to Dll activation
in the Mx primordium. However, their conclusion is supported by a delayed
pupal onset of Dll in ss- animals compared with normal
activation in late L3. We have not been able to observe this delay in
ss- mutants because with our culture conditions and
reference wild-type stock, we first detect Dll about 1-2 hours after the
L3/pupal stage (Fig. 4B). The most flagrant divergence between the antennal and maxillary programs resides in the timing for deployment of key signalling pathways and transcription factors: larval for the antenna and pupal for the maxillary organ. The central players common to the antennal and Mx programs show specific and divergent timelines as illustrated in Fig. 2. In the antennal territory, the Hh targets genes ptc, dpp and wg are activated co-temporally, whereas in the maxillary field dpp is delayed by roughly 12 hours, and wg for more than 2 days. The late creation of the dpp/wg interface presumably explains the late Dll and ss activation, and thus the delay in a clear morphological maxillary primordium until the beginning of the pupariation.
Temporal wg regulation is central to distinguishing Max/Ant identity
In the Mx field, we observed that the onsets of dpp (in L3 larvae)
and wg (in pre-pupae) were temporally uncoupled. Re-synchronising
wg and dpp in the Mx field by precociously expressing Wg
(Fig. 3) redirected Mx toward a
neo-antennal program deploying ss, dan, Dll and dac
(Fig. 4C,
Fig. 3A; not shown).
Conversely, Ss mis-expression leading to a Mx-to-antennal transformation is
also correlated with premature expression of Wg next to endogenous
dpp at the maxillary AP boundary
(Fig. 4D and not shown). Taken
together, these experiments thus indicate that the timing of Wg expression
coincides with the choice of primordial fate: maxillary versus antenna
(Fig. 3, model in
Fig. 6). We can conclude that
regulating temporal wg expression rather than Wg dose is crucial to
this decision, as Wg overexpression in pupae does not provoke a Mx-to-antennal
transformation (Fig. 3). Thus,
the developmental choice between two alternative identities is dependent upon
the time at which the instructive dpp/wg couple acts: larval
for antenna, later in pre-pupae for maxillary
(Fig. 2; model in
Fig. 6).
The delayed onset of wg expression in the Mx primordium presumably
depends on transcriptional regulation mediated by regulatory DNA elements. A
recent paper from Pereira et al. identified wg 3' flanking
regulatory sequences directing larval reporter gene expression in dorsal or in
ventral imaginal discs (Pereira et al.,
2006). None of their lines, including those that are expressed in
the larval antenna, showed detectable expression in the pupal maxillary
primordium (not shown). This indicates that maxillary directed wg
expression results from a spatiotemporal regulation distinct from the antenna.
A central landmark of the larval/pupal transition, the hormonal surge
triggering metamorphosis, might also be important in activating, or
de-repressing, maxillary wg expression. Ecdysone-responsive
cis-regulatory elements have been reported in the proneural gene
atonal required for sensory organ formation
(Niwa et al., 2004).
A decisive role for Wg in organ identity
The similar Mx-to-ant transformations induced by Wg or Ss indicate that
both are key players in distinguishing Mx from antennal identity. In normal Mx
development, the onsets of nuclear Ss selector protein and diffusible Wg
growth factor were temporally indissociable
(Fig. 4A). Our results from
loss-of-function experiments place wg signalling upstream of
ss in the Mx primordium, as abolishing wg signal
cell-autonomously silences ss
(Fig. 4G), whereas wg
is still expressed in the Mx primordium of a ss- mutant
(Fig. 4E). However, the
gain-of-function experiments show that ss can also activate
wg by an autoregulatory loop (Fig.
4D). This suggests that wg provides an obligatory input
in distinguishing Mx from Ant identities.
On confronting wggof (Mx-to-ant) with loss of
ss (stunted Mx), the resulting structures were unlike either
(Fig. 5A), suggesting that Ss
and Wg act at the same level. Importantly, mis-expressed Wg can reorganize the
Mx territory in the absence of Ss. In most cases, this yields an undefined
outgrowth (Fig. 5A3)
and occasionally gives rise to a distal clawed leg in place of the Mx palp
(Fig. 5A4). We infer
that the Mx-to-leg transformation seen for mis-expressed Wg in conjunction
with ss- reflects a Mx primordium that has already been
reoriented toward a pre-antennal environment by the action of Wg
(Fig. 5G). Accordingly, Dfd is
seen to retract from the maxillary field of wggof;
ss- larvae, as well as in the wggof alone
(Fig. 5E,C). Wg is thus unable
to activate Dan without ss but can confer antennal patterning
characteristics correlated with the absence of Dfd. Conversely, ss is
in any case sufficient to activate Dan, but without Wg it cannot induce Dfd
retraction from the Mx field (Fig.
5F,F'). Thus, the absence of Dfd is not a simple consequence
of Dan activation in the Mx field and reflects Wg activity. Taken together,
these various results indicate that Wg can contribute to a tissue
reorganisation involved in identity choice. Recent studies have implicated
regulation of signalling pathways as an important element in identity choice,
as dpp signalling regulated by Ultrabithorax in the haltere
and by vestigial in the wing helps distinguish between these
homologous structures (Crickmore and Mann,
2006; de Navas et al.,
2006; Makhijani et al.,
2007; Mohit et al.,
2006).
The present work demonstrates that the timing of wg expression is
involved in distinguishing maxillary from antennal organs. Wg is well known as
a potent mitogen, and we cannot exclude that its effect on
tissue-reorganisation and identity occurs at the level of cell proliferation
(Kenyon et al., 2003;
Serrano and O'Farrell, 1997).
Nevertheless, as wg exerts distinct effects on proliferation and
patterning in the developing wing (Neumann
and Cohen, 1996), these two aspects of wg function appear
to be separable. Similarly, Wg acts in specifying the wing primordium
independently of its DV axis specification
(Ng et al., 1996), and in
distinguishing the eye primordium from the dorsal head vertex
(Royet and Finkelstein, 1997).
The novel role of wg regulation in maxillary specification, where
changing the temporal framework for this single signalling output incites
maxillary cells to reorganise as an antennal organ, leads us to consider that
the Wg morphogen acts not only as a DV axial factor but also as an identity
determinant.
Appendage diversification
Appendage identity results from an interplay of regionalising signals and
selector transcription factors such as Hox genes. No Hox selector gene is
expressed in the antennal field, where identity has been attributed to the
instructive quality of co-expressed Dll and Hth
(Dong et al., 2000).
Downstream Hth/Dll targets include ss and its own targets,
dan and dan-related, which are required for antennal
differentiation (Emerald et al.,
2003; Suzanne et al.,
2003). The Mx primordium also possesses the configuration thought
to procure antennal identity: co-expression of Hth and Dll associated with Ss
expression in the Dll-expressing cells
(Fig. 2T,
Fig. 4B). However,
Ss-expressing cells of the antenna activate dan, whereas Mx cells
expressing Ss do not, raising the question what constitutes a cellular context
refractory to dan activation? One possible explanation would be that
the presence of Hox proteins in the Mx territory impedes dan
expression there. The Mx region expresses two Hox proteins, Dfd and Pb, which
might distinguish maxillary versus antennal identity. However,
pb-, Dfd- or double mutant pb-
Dfd- Mx primordia are not transformed to antenna and
dan is not expressed there (not shown). These Hox selectors are not
responsible for repressing dan expression in the Mx region, and thus
they do not control the crucial steps distinguishing maxillary versus antennal
programs. We propose a model where the pupal application of an antennal-like
program in the Mx primordium prevents ss-dependent dan
activation there. This hypothesis is supported by the fact that precociously
expressing Wg in the same territory is associated with premature Ss expression
that induces Dan there. That dan can be activated in larval maxillary
territory but not in pre-pupae suggests the existence of a larval competent
stage (Fig. 6, green
background) that is terminated in prepupae refractory to the same signal
(Fig. 6, yellow background).
Our working model supposes that Dfd contributes to elaborating Mx competence,
and proposes that the primary signal of Mx specification is the delayed Wg
expression in the prepupal stage (refractory to dan activation).
Conversely, earlier maxillary expression of Wg in the competent stage of
dan activation permits re-organisation toward antennal identity
(Fig. 6, Mx-to-Ant).
A driving force in metazoan evolution may have been the diversification of regulatory paradigms for controlling morphogen activities to create distinct appendages. The delayed initiation of Wg uncoupled from dpp in the maxillary primordium is an unexpected situation for a ventral appendage, and suggests that strategies for uncoupling dpp from wg may be important for diversifying developmental outcomes (Fig. 6). Our dissection of a novel program leading to a ventral appendage reveals that temporal regulation of signalling molecules may contribute to organ identity in as yet unexplored ways that help to create appendage diversity.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Abu-Shaar, M. and Mann, R. S. (1998). Generation of multiple antagonistic domains along the proximodistal axis during Drosophila leg development. Development 125,3821 -3830.[Abstract]
Agnes, F., Suzanne, M. and Noselli, S. (1999). The Drosophila JNK pathway controls the morphogenesis of imaginal discs during metamorphosis. Development 126,5453 -5462.[Abstract]
Belenkaya, T. Y., Han, C., Standley, H. J., Lin, X., Houston, D.
W., Heasman, J. and Lin, X. (2002). pygopus encodes a nuclear
protein essential for wingless/Wnt signaling.
Development 129,4089
-4101.
Benassayag, C., Plaza, S., Callaerts, P., Clements, J., Romeo,
Y., Gehring, W. J. and Cribbs, D. L. (2003). Evidence for a
direct functional antagonism of the selector genes proboscipedia and eyeless
in Drosophila head development. Development
130,575
-586.
Blackman, R. K., Sanicola, M., Raftery, L. A., Gillevet, T. and Gelbart, W. M. (1991). An extensive 3' cis-regulatory region directs the imaginal disk expression of decapentaplegic, a member of the TGF-beta family in Drosophila. Development 111,657 -666.[Abstract]
Brook, W. J., Diaz-Benjumea, F. J. and Cohen, S. M. (1996). Organizing spatial pattern in limb development. Annu. Rev. Cell Dev. Biol. 12,161 -180.[CrossRef][Medline]
Casares, F. and Mann, R. S. (1998). Control of antennal versus leg development in Drosophila. Nature 392,723 -726.[CrossRef][Medline]
Cohen, S. M. and Jurgens, G. (1989). Proximal-distal pattern formation in Drosophila: cell autonomous requirement for Distal-less gene activity in limb development. EMBO J. 8,2045 -2055.[Medline]
Cribbs, D. L., Benassayag, C., Randazzo, F. M. and Kaufman, T. C. (1995). Levels of homeotic protein function can determine developmental identity: evidence from low-level expression of the Drosophila homeotic gene proboscipedia under Hsp70 control. EMBO J. 14,767 -778.[Medline]
Crickmore, M. A. and Mann, R. S. (2006). Hox
control of organ size by regulation of morphogen production and mobility.
Science 313,63
-68.
de Navas, L. F., Garaulet, D. L. and Sanchez-Herrero, E.
(2006). The ultrabithorax Hox gene of Drosophila controls haltere
size by regulating the Dpp pathway. Development
133,4495
-4506.
Diaz-Benjumea, F. J., Cohen, B. and Cohen, S. M. (1994). Cell interaction between compartments establishes the proximal-distal axis of Drosophila legs. Nature 372,175 -179.[CrossRef][Medline]
Diederich, R. J., Pattatucci, A. M. and Kaufman, T. C. (1991). Developmental and evolutionary implications of labial, Deformed and engrailed expression in the Drosophila head. Development 113,273 -281.[Abstract]
Dominguez, M. and Casares, F. (2005). Organ specification-growth control connection: new in-sights from the Drosophila eye-antennal disc. Dev. Dyn. 232,673 -684.[CrossRef][Medline]
Dong, P. D., Chu, J. and Panganiban, G. (2000). Coexpression of the homeobox genes Distal-less and homothorax determines Drosophila antennal identity. Development 127,209 -216.[Abstract]
Dong, P. D., Chu, J. and Panganiban, G. (2001).
Proximodistal domain specification and interactions in developing Drosophila
appendages. Development
128,2365
-2372.
Dong, P. D., Dicks, J. S. and Panganiban, G. (2002). Distal-less and homothorax regulate multiple targets to pattern the Drosophila antenna. Development 129,1967 -1974.[Medline]
Duncan, D. M., Burgess, E. A. and Duncan, I.
(1998). Control of distal antennal identity and tarsal
development in Drosophila by spineless-aristapedia, a homolog of the mammalian
dioxin receptor. Genes Dev.
12,1290
-1303.
Emerald, B. S., Curtiss, J., Mlodzik, M. and Cohen, S. M.
(2003). Distal antenna and distal antenna related encode nuclear
proteins containing pipsqueak motifs involved in antenna development in
Drosophila. Development
130,1171
-1180.
Emmons, R. B., Duncan, D. and Duncan, I. (2007). Regulation of the Drosophila distal antennal determinant spineless. Dev. Biol. 302,412 -426.[CrossRef][Medline]
Gonzalez, F., Swales, L., Bejsovec, A., Skaer, H. and Martinez Arias, A. (1991). Secretion and movement of wingless protein in the epidermis of the Drosophila embryo. Mech. Dev. 35, 43-54.[CrossRef][Medline]
Haynie, J. L. and Bryant, P. J. (1986). Development of the eye-antenna imaginal disc and morphogenesis of the adult head in Drosophila melanogaster. J. Exp. Zool. 237,293 -308.[CrossRef][Medline]
Hinz, U., Giebel, B. and Campos-Ortega, J. A. (1994). The basic-helix-loop-helix domain of Drosophila lethal of scute protein is sufficient for proneural function and activates neurogenic genes. Cell 76,77 -87.[CrossRef][Medline]
Johnston, L. A. and Schubiger, G. (1996). Ectopic expression of wingless in imaginal discs interferes with decapentaplegic expression and alters cell determination. Development 122,3519 -3529.[Abstract]
Jurgens, G. and Hartenstein, V. (1993). The terminal regions of the body pattern. In The Development of Drosophila melanogaster (ed. M. Bate and A. Martinez Arias), pp.687 -746. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Kenyon, K. L., Ranade, S. S., Curtiss, J., Mlodzik, M. and Pignoni, F. (2003). Coordinating proliferation and tissue specification to promote regional identity in the Drosophila head. Dev. Cell 5,403 -414.[CrossRef][Medline]
Kumar, J. P. and Moses, K. (2001). EGF receptor and Notch signaling act upstream of Eyeless/Pax6 to control eye specification. Cell 104,687 -697.[CrossRef][Medline]
Lecuit, T. and Cohen, S. M. (1997). Proximal-distal axis formation in the Drosophila leg. Nature 388,139 -145.[CrossRef][Medline]
Makhijani, K., Kalyani, C., Srividya, T. and Shashidhara, L. S. (2007). Modulation of Decapentaplegic gradient during haltere specification in Drosophila. Dev. Biol. 302,243 -255.[CrossRef][Medline]
Merrill, V. K., Turner, F. R. and Kaufman, T. C. (1987). A genetic and developmental analysis of mutations in the Deformed locus in Drosophila melanogaster. Dev. Biol. 122,379 -395.[CrossRef][Medline]
Mohit, P., Makhijani, K., Madhavi, M. B., Bharathi, V., Lal, A., Sirdesai, G., Reddy, V. R., Ramesh, P., Kannan, R., Dhawan, J. et al. (2006). Modulation of AP and DV signaling pathways by the homeotic gene Ultrabithorax during haltere development in Drosophila. Dev. Biol. 291,356 -367.[CrossRef][Medline]
Morata, G. (2001). How Drosophila appendages develop. Nat. Rev. Mol. Cell Biol. 2, 89-97.[CrossRef][Medline]
Morata, G. and Lawrence, P. A. (1979). Development of the eye-antenna imaginal disc of Drosophila. Dev. Biol. 70,355 -371.[CrossRef][Medline]
Neumann, C. J. and Cohen, S. M. (1996). Distinct mitogenic and cell fate specification functions of wingless in different regions of the wing. Development 122,1781 -1789.[Abstract]
Ng, M., Diaz-Benjumea, F. J., Vincent, J. P., Wu, J. and Cohen, S. M. (1996). Specification of the wing by localized expression of wingless protein. Nature 381,316 -318.[CrossRef][Medline]
Niwa, N., Hiromi, Y. and Okabe, M. (2004). A conserved developmental program for sensory organ formation in Drosophila melanogaster. Nat. Genet. 36,293 -297.[CrossRef][Medline]
Panganiban, G. (2000). Distal-less function during Drosophila appendage and sense organ development. Dev. Dyn. 218,554 -562.[CrossRef][Medline]
Pereira, P. S., Pinho, S., Johnson, K., Couso, J. P. and Casares, F. (2006). A 3' cis-regulatory region controls wingless expression in the Drosophila eye and leg primordia. Dev. Dyn. 235,225 -234.[CrossRef][Medline]
Pultz, M. A., Diederich, R. J., Cribbs, D. L. and Kaufman, T.
C. (1988). The proboscipedia locus of the Antennapedia
complex: a molecular and genetic analysis. Genes Dev.
2, 901-920.
Royet, J. and Finkelstein, R. (1997). Establishing primordia in the Drosophila eye-antennal imaginal disc: the roles of decapentaplegic, wingless and hedgehog. Development 124,4793 -4800.[Abstract]
Serrano, N. and O'Farrell, P. H. (1997). Limb morphogenesis: connections between patterning and growth. Curr. Biol. 7,R186 -R195.[CrossRef][Medline]
Struhl, G. (1982). Spineless-aristapedia: a
homeotic gene that does not control the development of specific compartments
in Drosophila. Genetics
102,737
-749.
Suzanne, M., Estella, C., Calleja, M. and Sanchez-Herrero, E. (2003). The hernandez and fernandez genes of Drosophila specify eye and antenna. Dev. Biol. 260,465 -483.[CrossRef][Medline]
Wilder, E. L. and Perrimon, N. (1995). Dual functions of wingless in the Drosophila leg imaginal disc. Development 121,477 -488.[Abstract]
Wu, J. and Cohen, S. M. (1999). Proximodistal axis formation in the Drosophila leg: subdivision into proximal and distal domains by Homothorax and Distal-less. Development 126,109 -117.[Abstract]
Xu, T. and Rubin, G. M. (1993). Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117,1223 -1237.[Abstract]
Zhang, Y. and Kalderon, D. (2000). Regulation of cell proliferation and patterning in Drosophila oogenesis by Hedgehog signaling. Development 127,2165 -2176.[Abstract]
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