1 Centro de Biologia Molecular `Severo Ochoa', C.S.I.C., Universidad Autonoma de
Madrid, Cantoblanco, E-28049 Madrid, Spain
2 Centro de Investigaciones Biológicas, C.S.I.C., Velázquez 144,
28006 Madrid, Spain
* Author for correspondence (e-mail: iguerrero{at}cbm.uam.es)
Accepted 18 October 2002
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SUMMARY |
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Key words: Drosophila genital disc patterning, Hh signalling, Dpp signalling, Wg signalling, Segmental boundaries, Cell lineage restriction
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INTRODUCTION |
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The genital disc, which gives rise to the terminalia of the adult fly
(internal and external genitalia excluding the gonads and analia)
(Bryant, 1978), is organised
in a manner reminiscent of the leg disc
(Gorfinkiel et al., 1999
;
Estrada and Sánchez-Herrero,
2001
). However, there are several differences between the genital
disc and other imaginal discs that make it an original experimental model
(reviewed by Sánchez and Guerrero,
2001
). For example, it is the only imaginal disc that shows clear
sexual dimorphism. The genital disc is a compound disc formed by the fusion of
three different primordia derived from the embryonic abdominal segments 8, 9
and 10-11 (Nöthiger et al.,
1977
; Schüpbach et al.,
1978
; Dübendorfer and
Nöthiger, 1982
; Epper and
Nöthiger, 1982
). Thus, a genital disc contains a female
genital primordium (derived from segment A8), a male genital primordium
(derived from segment A9) and an anal primordium (derived from segments
A10-11). In females, the female primordium gives rise to almost all the
internal and external genitalia. The male primordium was considered to be a
`repressed primordium' because its growth is limited and it does not form any
adult structures (Epper and Nöthiger,
1982
). However, it has recently been shown that the male
primordium in the female genital disc gives rise to the parovaria (internal
accessory female glands) (Keisman et al.,
2001
). The anal primordium in females gives rise to the female
analia. The converse is true for males. In male genital discs, the male
genital primordium gives rise to the majority of the internal and external
male genital structures. The female genital primordium was also considered to
be a repressed primordium but it is now known to produce a miniature eighth
tergite (Keisman et al.,
2001
). The anal primordium in the male genital disc gives rise to
the characteristic male anal plates.
Contrary to the situation in other imaginal discs, in the genital disc, the
response to Hh, Dpp and Wg is controlled by the sex determination genes
(Sánchez et al., 2001;
Keisman and Baker, 2001
). The
last gene in the sex determination cascade is doublesex
(dsx), which encodes two zinc finger proteins, DsxM in
males and DsxF in females. In the female genital discs,
DsxF blocks the transcription of dpp induced by Hh in the
A9, while in the male genital disc, DsxM modulates the response to
Wg in A8 cells. This results in the differential growth and development of the
genital primordium of the corresponding sex and its respective `repressed
primordium'.
Apart from the additional control exerted by the sex determination genes in
the genital disc, the fact that three different segments develop side by side
is a further characteristic feature of genital discs that distinguishes them
from other imaginal discs. Each primordium of the genital disc has an anterior
(A) and a posterior (P) compartment
(Freeland and Kuhn, 1996;
Casares et al., 1997
;
Chen and Baker, 1997
). The
situation is thus similar to that in the embryo and in the abdomen, where P
cells from one segment come into contact with A cells located anteriorly at
the AP compartment border, and with A cells located posteriorly at the PA
segmental border. Extensive work on the abdomen has shown that Hh signals to A
cells both for and aft, and that these cells respond differently to the Hh
signal (Struhl et al., 1997a
;
Lawrence et al., 1999
). Hh
induces optomotor-blind (omb) expression in cells at the AP
border but not at the segmental border
(Kopp and Duncan, 1997
). In
the embryo, Hh induces Wg expression in cells at the AP border, while no Wg
expression is observed at the PA border
(O'Keefe et al., 1997
;
Sanson et al., 1999
).
It has been shown that both AP and PA boundaries act as cell lineage
restriction borders, although in the embryo, PA restriction occurs later than
the AP restriction (Vincent and O'Farrell,
1992). Cell lineage restriction studies have shown that an
interface between En/Hh and non-En/Hh expressing cells is required to form
such boundaries (García-Bellido et
al., 1973
; Kornberg,
1981
; Rodríguez and
Basler, 1997
; Blair and
Ralston, 1997
). In the genital disc, a cell lineage restriction
border was found between the genitalia and analia, indicating that a segmental
boundary exist between A8/A9 and A10-11
(Dübendorfer and Nöthiger,
1982
). It has also been shown that by the beginning of the second
instar, or even earlier, the genital disc has already subdivided into anterior
and posterior compartments (Chen and
Baker, 1997
).
We have reanalysed the presence of cell lineage restriction borders in the genital disc and address the question of whether segmental borders (the borders between the different primordia) differ from compartment borders. We have also looked for communication between the different primordia and explored whether this communication is required for the development of the genital disc. The results obtained prompt our reconsideration of genital disc organisation, and we propose a situation in which the three primordia develop interdependently.
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MATERIALS AND METHODS |
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Flies used for the analysis of external terminalia of adults were kept in a mixture of ethanol:glycerol (3:1) for several days. They were then macerated in 10% KOH at 60°C for 15 minutes, thoroughly washed with H2O, and mounted in Faure's solution for examination under a compound microscope.
Clonal analysis
Clones of mutant cells were generated by FLP-mediated mitotic recombination
(Golic, 1991) and lineage
clones were generated using the flip-out GAL4 system
(Pignoni and Zipursky, 1997
).
For the generation of smo and smo cad clones, yFLP122;
smo FRT 40, hhlacZ or yFLP122; smo cad FRT40 females were
crossed to GFP FRT40 males and 24-72 hour larvae were heat shocked
for 60-90 minutes. For the generation of lineage clones in a
dsx1 background, act>>lacZ;
dsx1/SM5;TM6B/+ females were crossed to yFLP122;
dsx1/SM5, TM6B/+ males and 12-24 hour embryos were
heat-shocked for 10 minutes. Lineage clones were also generated crossing
act>>GAL4 UAS-GFP females with HmcFLPIII males and
12-24 hour embryos were heat-shocked for 10 minutes.
Antibody staining
The antibodies used were the following: monoclonal anti-En 4D9
(Patel et al., 1989);
polyclonal anti-Cad (Macdonald and Struhl,
1986
); anti-Ptc antibody ApaI
(Capdevila et al., 1994
);
monoclonal anti Abd-B 1A2E (Celniker et
al., 1989
); monoclonal anti-Wg
(Brook and Cohen, 1996
);
anti-Tsh antibody (S. Kerridge); anti-Dll antibody
(Vachon et al., 1992
);
monoclonal anti-MYC 9E10 (Babco, Berkeley antibody company); and
anti-ß-gal (Jackson Laboratories).
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RESULTS |
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In male genital discs, the A8 segment is a small mass of cells located at the ventral posterior end of the disc (Fig. 1) and corresponds to the `female repressed primordium' (RFP) with A and P compartments, the latter being contiguous to the A compartment of the A9 segment (the male genital primordium, MGP). We also observed two bands of Ptc expression flanking the P cells of the small A8 segment in male genital discs, one corresponding to the anterior cells of the A8 (Fig. 2A, arrowhead), and the other, to a narrow band of cells in A9 (Fig. 2A, arrow).
In female genital discs, the border between segments A8 and A9 was more difficult to detect because of complex folding of the disc. We were unable to distinguish two different bands of Ptc expression flanking the A8 posterior compartment, probably because the new band of Ptc expression induced in A9 by posterior A8 cells fuses with the `normal' Ptc-expressing cells at the A9 AP compartment border (Fig. 2C).
These observations led us to question whether anterior cells respond
differently to the Hh signal depending on their position with respect to the
Hh source, as occurs in the embryo and abdomen
(Heemskerk and DiNardo, 1994;
O'Keefe et al., 1997
;
Struhl et al., 1997a
;
Alexandre et al., 1999
). To
address this question, we concentrated on analysing the segmental border
between segments A9 (male genital primordium) and A10-11 (anal primordium),
owing to its improved visibility over the A8/A9 border, both in male and
female genital discs. As previously shown, in male genital discs Hh signals
anterior A9 cells, inducing the activation of Wg and Dpp in mutually exclusive
domains (Chen and Baker, 1997
;
Sánchez et al., 1997
;
Gorfinkiel et al., 1999
). In
female genital discs, anterior A9 cells respond to Hh signal, activating Wg
only; Dpp activation was not observed because the sex-determination gene
DsxF blocks its transcription in response to Hh
(Sánchez et al., 2001
).
We then went on to determine how the anterior A10-11 cells responded to Hh
emerging from posterior A9 cells. No expression of Wg or Dpp was observed
(data not shown).
We have observed that en and hh expression domains did
not fully coincide in posterior A9 cells
(Fig. 3A). At the anterior
border of the A9 posterior compartment, En and hh-lacZ colocalised
and their limit of expression appeared as a straight edge. By contrast, at the
posterior border of A9, the limits were more diffuse, and the En expression
domain extended beyond the hh-lacZ domain in the anterior region of
segments A10-11 (Fig. 3B). To
establish whether the segmental border corresponds to the posterior border of
the Hh expression domain or to that of En expression, we stained genital discs
for Cad and En. Surprisingly, we found that there was a narrow band of cells
where Cad and En overlapped (Fig.
3C,D). This indicated that En was expressed in anterior A10-11
cells. This situation resembles that of the AP compartment border of wing
imaginal discs, where En is expressed in anterior cells
(Blair, 1992;
Hidalgo, 1994
) in response to
Hh (Guillén et al.,
1995
; de Celis and Ruiz-Gomez,
1995
) in late third instar larval imaginal discs. We also noted
that the limit of Cad expression was not well defined. On the contrary, Cad
levels were downregulated at the A9/A10-11 border
(Fig. 3D). To determine if
these properties of the A9/A10-11 border (i.e. Ptc expression, En expression
and Cad downregulation) were due to a response to the Hh signal, we
experimentally altered Hh levels. To increase Hh levels, we analysed genital
discs from en-GAL4/UAS-hh flies. In these discs, we observed expanded
Ptc and En expression domains in anterior A10-11 cells abutting the A9/A10-11
segmental border (Fig. 4A).
Consistently, the region where Cad levels were downregulated was also extended
(Fig. 4B).
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|
To test if Hh signal crosses the segmental border to adjacent A cells of
the following segment, homozygous null clones for smoothened
(smo) were induced. Smo is a serpentine transmembrane protein
required for Hh signal transduction (Alcedo
et al., 1996; van den Heuvel
and Ingham, 1996
). In smo- clones, Hh
signalling is abolished (Chen and Struhl,
1996
). Accordingly, smo- clones in A10-11 that
abut A9 eliminated both Ptc (Fig.
4C) and En expression (Fig.
4D), indicating that En in the A10-11 was induced by Hh as occurs
in the wing imaginal disc (Blair,
1992
).
These results show that cells located anteriorly and posteriorly to the Hh source respond differently. While anterior A9 cells respond to Hh by activating Wg and/or Dpp, anterior A10-11 cells respond by activating En expression. The fact that Hh signals both infront and behind leads us to the issue of whether the segmental borders behave as compartment borders with respect to cell lineage restriction.
Cell lineage restriction among genital disc primordia
Cell lineage restriction borders are formed at the interface between En/Hh
and non-En/Hh expressing cells (reviewed by
Dahmann and Basler, 1999).
Hence, we tried to precisely determine the domains of En expression in the
genital disc. This is not straightforward because of the complex folding of
the disc. Carefully observation of En and Hh expression in male and female
genital discs showed the three P compartments to be contiguous at the lateral
edges of the disc. This was best observed in the A9 and A10-11 segments
(Fig. 3A). Note that En
expression is continued over the male genital primordium and the anal
primordium (schematised in Fig.
1).
In the light of these observations, we re-analysed the behaviour of lineage clones. We first set out to determine whether the border between male and female genitalia constitutes a cell lineage restriction border. Secondly, we wanted to determine if Hh signalling, as in compartment borders, establishes the segmental borders.
To address the first question, we performed a clonal analysis in
dsx1/dsx1 genital discs, where both genital
primordia grow at a similar rate and produce intersexual adult structures (P.
C. Ehrensperger, PhD thesis, University of Zürich, Switzerland, 1983).
Dübendorfer and Nöthiger
(Dübendorfer and Nöthiger,
1982) have previously reported the existence of two separate cell
lineages for the genitalia and analia in the developing genital discs.
However, no cell lineage restriction between the male and female genitalia
could be determined, as these authors were unable to detect any adult
structures developing from the `repressed primordia'. We induced ß-gal
clones in this mutant combination, between 0 and 24 hours of development, and
monitored the clones in third instar larval genital discs. To distinguish the
segmental border between male and female genital primordia, we took advantage
of the specific expression of teashirt (tsh) in the A8
segment, thus labelling the female genital primordium
(Fig. 5A). We observed that
ß-gal clones remained confined to either the male or female genital
primordia, indicating the existence of a cell lineage restriction barrier
between both primordia (Fig.
5A). Similarly, ß-gal clones in the male genital primordium
did not cross towards the anal primordium
(Fig. 5B).
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It has been shown that at the AP compartment border of wing imaginal discs,
A cells that lack smo and abut the AP border change their affinity
and no longer mix with surrounding anterior cells but instead integrate with P
cells. These cells were identified as clones of cells in the P compartment
lacking Hh expression (thus of anterior identity)
(Blair and Ralston, 1997;
Rodríguez and Basler,
1997
). To determine if the cell lineage restriction properties at
the segmental border also depend on Hh signalling, we analysed the behaviour
of smo- clones in the area around the border between A9
and A10-11 segments. Fig. 6A
shows that smo- clones in the anterior compartment of
A10-11 that abut the compartment border behave as described, i.e. they easily
integrate among the P cells of the corresponding segment (detected as a clone
of cells showing no hh-lacZ expression), while the twin clone remains
at the A compartment (Fig. 6A,
clone 3). However, smo- clones in the anterior compartment
of A10-11 that abut the segmental border did not integrate with P cells of the
A9 (male genital primordium). Instead, both the clone and the twin remained in
the anterior A10-11 (Fig. 6B,
clone 4). This result indicates that, besides Hh, some other factor was
required to maintain the differential cell affinities at the segmental
borders. Altogether, these findings suggest the segmental border does not
behave as a compartment border, where the lineage properties of the cells are
mainly dependent on Hh function. Instead, it seems that the segmental border
between A9 (male genital primordium) and A10-11 (anal primordium) also depends
on factors other than Hh and En.
|
The Hox genes are factors that might be involved in defining cell lineage
restriction properties. These genes have segmental expression boundaries in
the genital disc. Two Hox genes [Abdominal B (Abd-Bm and
Abd-Br)] and cad are specifically expressed in the genital
disc: Abd-Bm in A8, Abd-Br in A9 and cad in the
A10-11 segments. It has been shown that Abd B-m and r
specify the female and male genitalia
(Estrada and Sánchez-Herrero,
2001) and cad specifies the analia
(Moreno and Morata, 1999
), and
that cad lack of function clones autonomously activate Abd-B
in the analia (Moreno and Morata,
1999
). We thus explored the possibility that double mutant clones
for smo and cad might be able to straddle the boundary
between A9 and A10-11. smo and cad double mutant clones were
incapable of straddling the segmental border between A9 and A10-11
(Fig. 6C, clones 5 and 6),
suggesting that this segmental border is not dependent on the Hox genes.
Diffusion of morphogenetic signals across segmental borders
To determine whether there is a segmental restriction to the diffusion of
morphogenetic signals in the genital disc, we ectopically expressed Hh in the
analia, and examined its effect in the genitalia.
cad-GAL4/UAS-hh females and males showed the complete
duplication of external genital structures
(Fig. 7C,D) and accordingly,
duplication of the genital discs (Fig.
7E,F). These genital duplications were not produced when we
autonomously activated the Hh pathway in the analia with a dominant negative
form of ptc (ptcSSD)
(Martín et al., 2001)
(data not shown). These results indicate that Hh from the analia is able to
specify positional information in the more anterior segments, that is, in the
genitalia. Spread of the Hh signal can occur through the common P
compartments, as en-GAL4/UAS-hh also induced the same type
of genital duplications (data not shown). We then tested whether the other
morphogenetic signals, Wg and Dpp, were also able to diffuse across the
segmental border between analia and genitalia.
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Females of the mutant combination
dppd5/dppd12 had normal vaginal plates and
tergite eight, showing only discretely reduced analia in a few cases. On the
contrary, males displayed extensively reduced external terminalia
(Fig. 8A), only presenting
structures of the penis apparatus that could be duplicated or even
triplicated. The development of the penis apparatus depends mainly on Wg
(Sánchez et al., 1997),
and due to the mutual repression between wg and dpp, Wg is
expanded in a dpp mutant background, resulting in the duplications of
the penis apparatus observed. To test the ability of Dpp to diffuse across the
segmental border, we induced ectopic Dpp clones in a
dppd5/dppd12 mutant background. These clones
always led to the recovery of the terminal structures
(Fig. 8B). This recovery
depended on the developmental stage at which the clone was induced. Thus, the
earlier the clone was induced, the greater the inventory of structures
differentiated by the genital disc. Recovery was accompanied by reduced penis
apparatus structure duplications, and could be associated with duplications
that mainly comprised the genital arch or lateral plate, two structures that
require dpp activity
(Sánchez et al., 1997
).
A property of all clones was their non-autonomous character: the
Dpp-expressing clones marked with yellow always induced the
development of unmarked structures. In some cases, the yellow
bristles were only found in the analia
(Fig. 8B), whereas in others
they were found in the genitalia (data not shown). In either case, the
corresponding genitalia and analia were also recovered. This non-autonomous
effect of Dpp indicates that there is signal diffusion between the genital and
the anal primordia, despite the fact that they belong to different
segments.
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To further test the ability of Dpp to spread through the genital-anal
border, we specifically expressed Dpp in the analia in
cad-GAL4/UAS-dpp flies and monitored effects in the genitalia.
Complete duplication of the vaginal plates was observed (data not shown), in
agreement with the effect induced by ectopic Hh. However, in some cases, all
the terminalia were deleted (data not shown). Next, we tried to establish if
the ability of ectopic Dpp in the analia to duplicate genital structures was
due to Dpp itself or to downstream signals activated by Dpp. We therefore
tested the effect of the ectopic expression of a constitutive active form of a
Dpp receptor, thickvein (tkv). The expression of this gene
autonomously activates the Dpp pathway
(Lecuit et al., 1996;
Nellen et al., 1996
). We found
that cad-GAL4/UAS-tkv flies always lacked both analia and genitalia
(data not shown). This indicates that the duplications obtained were not
caused by a secondary signal activated by Dpp. Taken together these results
indicate that Dpp is able to spread between the analia and genitalia.
Females flies of the wg mutant combination
wgCX3/Sp (Sp is also a wg allele)
(Neumann and Cohen, 1996) have
reduced vaginal plates and tergite eight, and the vulva is mainly absent
(Fig. 8C). Mutant males have a
strong phenotype in the genitalia: the penis apparatus and most of the clasper
are absent and the lateral plates are fused
(Fig. 8E). To analyse further
the role of Wg in genital disc development as well as the non-autonomous
effect of Wg, we overexpressed wg in the anal primordium in
cad-GAL4/UAS-wg; wgCX3/Sp flies. Some of the
females showed recovery of the vaginal plates and tergite eight but not the
vulva. The analia were much reduced in some cases
(Fig. 8D). Other females showed
no genital and anal structures. Males showed recovery of genital structures
such as claspers and penis apparatus, although this latter structure was
sometimes still reduced (Fig.
8F). Analia were almost normal or reduced. Thus, the ectopic
expression of Wg in the analia can either recover structures of the genitalia
or can prevent the development of both genitalia and analia.
To further explore the ability of Wg to spread through the genital-anal
border, we specifically expressed Wg in the analia in cad-GAL4/UAS-wg
flies, and monitored the effect on the genitalia. Small duplications of the
penis apparatus or the complete absence of the terminalia were observed. We
then overexpressed the downstream components of the Wg pathway Tcf
(van de Wetering et al., 1997;
Brunner et al., 1997
;
Riese et al., 1997
) and a form
of Armadillo (Arm) that is constitutively active
(Zecca et al., 1996
;
van de Wetering et al., 1997
).
In both cases, the phenotype observed was the same as when Wg was ectopically
expressed. Interestingly, diffusion of ectopic Wg protein from the analia
towards the genitalia was not observed and the excess of Wg protein was
confined to the analia of cad-GAL4/UAS-wgts genital discs
(data not shown). This is in agreement with the observation that
en-expressing cells make a barrier to the diffusion of Wg in the
embryonic segment (Dubois et al.,
2001
) (see Discussion). These results suggest that a signal
activated by Wg is responsible for the non-autonomous effect that the
expression of this gene in the analia has in the genitalia.
In the experiments described above, we sometimes found that high level of
Wg and Dpp in the analia impeded the development of both analia and genitalia.
This suggested that the analia might be required for the development of the
whole genital disc. To test this possibility, we induced ablation of the anal
primordium cells using the toxic UAS-ricin A transgene that it has
been shown to be efficient, cell specific and cell autonomous
(Hidalgo et al., 1995;
Hidalgo and Brand, 1997
).
Ricin A is the catalytic subunit of Ricin toxin, which is
capable of killing a cell but not of crossing the cell membrane into
neighbouring cells. In the form employed for transgenic expression in flies,
Ricin lacks the subunit B and a secretory signal peptide, necessary
for toxin internalisation and spreading to neighbouring cells
(Moffat et al., 1992
).
Ricin A chain kills cells by depurinating 28S ribosomal RNA and
effectively halting protein synthesis. We have observed that
cad-GAL4/UAS-ricin A flies lack all the terminal structures
(Fig. 9A,B). Dissection of
these larvae at late third instar showed that the genital discs had not
proliferated (Fig. 9C).
|
Next, to see the effect of ablation of cells of the genital primordia in
the development of the genital disc, we expressed Ricin protein in the
repressed female genital primordium (A8) of male genital discs by using
tsh-GAL4. As tsh is expressed up to A9 segment during
embryonic development, to avoid lethality of the embryos and allow them to
develop into third larval stage so that the genital disc could be analysed, a
cold-sensitive Ricin protein was used
(Moffat et al., 1992). It was
observed that inducing the functional Ricin protein at the second larval
instar causes a reduction of proliferation of the genital disc
(Fig. 9E) compared with a
genital disc where the Ricin protein was non-functional during whole
development (Fig. 9D). Note
also that Distal-less expression was absent in the male genital primordium
(A9) of the male genital disc that showed reduced proliferation
(Fig. 9E).
Collectively, these results suggest that cell communication between the different primordia that form the genital disc is required for the development of all terminalia.
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DISCUSSION |
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Interactions among segmental primordia are required for genital disc
development
The three segmental primordia of the genital disc are contiguous. This
means that the P compartment of one primordium is adjacent to A cells of the
corresponding primordium and to A cells of the following primordium. In
addition, the P compartment cells of the three segments converge in lateral
areas of the genital disc (Keisman and
Baker, 2001) (this paper). Hh is expressed in the three P
compartments (Freeland and Kuhn,
1996
; Casares et al.,
1997
; Chen and Baker,
1997
). It is shown here, that Hh activates target genes in the
receiving cells both behind and infront. These target genes are different on
each side of its expression domain. Particularly, Hh at the posterior
compartment of the male genital primordium (A9 segment) signals anteriorly,
inducing Wg and/or Dpp expression in anterior cells of this primordium, and
posteriorly, inducing Ptc expression. Hh also posteriorly signals anterior
cells of the anal primordium (segments A10-11) inducing En expression in a
narrow band of cells. Interestingly, Cad expression is reduced in these cells.
A similar situation has been described in embryonic segments in which Hh
activates wg at the AP border and rhomboid at the segmental
border (O'Keefe et al., 1997
;
Alexandre et al., 1999
). Hh
controls Wg and EGF signalling pathways on each side of its expression domain
in embryos. We therefore expected to find that, at the segmental border, Hh
would activate a target gene implicated in a signalling mechanism. We tested
specific expression at the segmental border of members of several signalling
pathways, but unfortunately no such expression pattern was observed.
Hh has a pivotal role in the morphogenesis of all imaginal discs. Ectopic
Hh gives rise to duplications of parts of, or whole, appendages in the
imaginal discs of the fly (reviewed by
Lawrence and Struhl, 1996). In
wild-type genital discs, such as in the leg and antenna, Hh induces the
expression of wg and dpp in A compartment cells close to the
AP border of each of the three primordia. It is shown here, that the ectopic
expression of hh in the anal primordium induces complete duplication
of the genital disc with the corresponding expression of these genes in their
normal expression domains. The repressed male and female primordia also seemed
to be duplicated in the female and male genital discs, respectively. These
results indicate again that Hh diffuses across the border between the
genitalia and analia, although this border acts as a cell lineage restriction
barrier (see below). It should be noted that Hh also diffuses across the
border between the embryonic segments
(Heemskerk and DiNardo, 1994
)
and the abdominal segments of adult flies
(Struhl et al., 1997a
;
Struhl et al., 1997b
;
Lawrence et al., 1999
). The
results presented here also show that the ectopic expression of either
dpp or wg in the analia also affects the development of the
male and female genitalia.
The non-autonomous effect that ectopic Dpp in the analia has on the development of the genitalia is due to diffusion of Dpp itself from the analia to the genitalia, and not to the non-autonomous effect of Dpp downstream genes. By contrast, the same effect on the development of the genitalia was observed when Wg itself or any of the downstream components of the Wg-pathway, Tcf or Arm, were ectopically expressed in the analia. These results together with the observation that no Wg protein was detected in the genitalia when it was ectopically expressed in the analia indicate that the non-autonomous effect of ectopic Wg is due to an unknown signal activated by the Wg-pathway.
It has been recently described that Wg spreads and acts within the
embryonic epidermis of Drosophila in different ranges in anterior and
posterior directions (Sanson et al.,
1999; Sanson,
2001
; Dubois et al.,
2001
). Transport or stability is reduced in
engrailed-expressing cells, and further posterior Wg movement is
blocked at the presumptive segmental boundary. hh function is
involved in the formation of this barrier. If Wg diffusion across the
genitalia-analia border is limited, it might be established very early in
development by a similar mechanism to that observed in the embryo.
The lack of development of the analia gives rise to a lack of genital
structures, consistent with the outcome of genetic ablation of the analia by
Ricin A (see above). This result indicates that morphogenetic signals diffuse
from the analia to the genitalia, and that this diffusion is needed for the
normal development of the genital disc. However, does diffusion also occur
between the male and female genital primordia of the genital disc? The results
obtained in the clonal analysis of transformer (tra) in the
female genital disc are relevant to this question
(Wieschaus and Nöthiger,
1982). It was found that male tra- clones
could give rise to male genital structures associated with a loss of female
genital structures (vaginal plates and tergite eight). These data suggest a
communication system between both genital primordia. Results presented here
support this contention. Ablation of cells of the repressed female genital
primordium of a male genital disc by Ricin protein causes a reduction of
proliferation of the genital disc.
In summary, the development of the genital disc, as that of other imaginal discs, requires interaction among the compartments forming each of its three primordia. The results presented here indicate that cell communication among different segmental primordia is also required for the development of the genital disc.
Cell lineage among genital disc primordia
A study of cell lineage in the male and female genital discs revealed that
there is a cell lineage restriction between the analia and the female or the
male genitalia (Dübendorfer and
Nöthiger, 1982). No cell lineage restriction between the male
and female genitalia could be determined as each of these does not develop in
the opposite sex and consequently does not produce adult structures. For this
reason, we performed a clonal analysis in intersexual flies in which both
genital primordia develop. We found that there is a cell lineage restriction
barrier between female (A8) and male (A9) primordia, and between male and anal
(A10-11) primordia. Even though the P compartment cells of the three segments
converge in the lateral areas of the disc, there is still a cell lineage
restriction among the P cells of different segments. In other imaginal discs,
such as those of the wing and leg discs composed of an A and P
compartment the cell lineage restriction between A and P cells is a
consequence of the different affinity of posterior En/Hh expressing cells and
anterior non-En/Hh expressing cells.
The present results indicate that Hh signalling is not responsible for the
cell lineage restriction between segmental primordia between A9 and A10-11.
The question arises as to how this cell lineage restriction is achieved. We
found that the Hox gene cad, which is expressed only in the anal
primordium (Moreno and Morata,
1999), is not required for the restriction at the segmental border
between the male genital primordium (A9) and anal primordium (A10-11). The
gene Abd-B, which produces two different Abd-Bm and Abd-Br proteins
(Casanova et al., 1986
;
DeLorenzi et al., 1988
;
Kuziora and McGinnis, 1988
;
Sánchez-Herrero and Crosby,
1988
), is expressed in the genital primordia. Abd-Bm is only
present in the female genital primordium (A8), whereas Abd-Br is only found in
the male genital primordium (A9) (Casares
et al., 1997
; Estrada and
Sánchez-Herrero, 2001
). This suggests that the Hox genes
might not only have a role in defining the identity of each segment of the
genital disc (Estrada and
Sánchez-Herrero, 2001
) but may also be involved in
establishing the cell lineage restriction among segmental boundaries. However,
we found that smo; cad double mutant clones were still unable to
straddle the segmental boundary between the A9 and A10-11 segments. Moreover,
we observed the overlapping of Abd-B and Cad expression domains at the border
between A9 and A10-A11 (data not shown). Thus, this segmental boundary does
not behave like other segmental boundaries where the Hox expression domains
are exclusive. Nevertheless, it is still possible that the interface between
Abd-Bm and Abd-Br expressing cells forms the A8-A9 lineage restriction
barrier.
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ACKNOWLEDGMENTS |
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