Centro de Biología Molecular CSIC-UAM, Universidad Autónoma de Madrid, 28049 Madrid, Spain
* Author for correspondence (e-mail: nazpiazu{at}cbm.uam.es)
Accepted 3 June 2003
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SUMMARY |
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Key words: eyg, toe, Thorax, Pax genes, Drosophila
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INTRODUCTION |
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However, within each segment, the morphological diversity does not depend
on the Hox genes but on other developmental genes that are expressed in
specific regions within segments. For example, engrailed (en)
determines the difference between A and P compartments
(Morata and Lawrence, 1975),
apterous (ap) is involved in distinguishing the dorsal and ventral
regions of the wing (Diaz-Benjumea and
Cohen, 1993
), and pannier (pnr) discriminates the
dorsal-medial and dorsal-lateral regions of the thorax and abdomen
(Calleja et al., 2000
;
Herranz and Morata, 2001
).
The second thoracic segment develops in the dorsal region of the wing and
the corresponding part of the thorax, known as mesothorax. These structures
derive from the wing imaginal disc, which is subdivided from the beginning
into A and P compartments (Lawrence and
Morata, 1977). This subdivision affects both the wing and the
thoracic region. In the mesothorax, the A compartment forms the greater part,
the notum, whereas the P compartment is a small featureless region known as
the postnotum.
The notum is made up of approximately 15,000 cells that develop a complex
and highly stereotyped bristle pattern in which no lineage restriction has
been found (Calleja et al.,
2000). Thus, its genetic/morphological diversity has to be
generated by non-lineage subdivisions. The notum has been shown to be
subdivided into two major regions defined by the activities of the
pnr and Iroquois (Iro) genes
(Calleja et al., 2000
;
Gomez-Skarmeta et al., 1996
).
The Iro genes are expressed originally in all the notum cells and specify the
development of the entire notum (Diez del
Corral et al., 1999
). Later, pnr restricts Iro gene
activity to the lateral region and also specifies dorsal-medial development
(Calleja et al., 2000
).
However, pnr and the Iro genes are expressed in comparable domains
in head, thoracic and abdominal segments, suggesting that they encode general
properties such as dorsal-medial or dorsal-lateral, which apply to all
segments. That is, they do not determine the development of one specific
segment, but are probably involved in a general combinatorial mechanism
together with other general factors, such as the Hox genes or
engrailed (reviewed by Mann and
Morata, 2000). It follows that there should exist other genes,
regulated by the combinations of the above selector genes, which would be
responsible for the morphological diversity within the different segments.
Using the `yellow' method (Calleja et
al., 1996), we have isolated a number of Gal4 lines conferring
expression in various parts of the thorax. One of them (EM461) yielded Gal4
activity in most of the scutum, the part of the notum from the anterior border
to the suture with the scutellum (Fig.
1). No activity was observed in the abdomen. We report a
functional study of a gene whose expression directs Gal4 activity in the EM461
line. The gene was discovered to be eyegone (eyg), which encodes a
homeodomain Pax protein (Jun and Desplan,
1996
). eyg is one of the elements of the genetic network
activated during eye development (Hazelett
et al., 1998
; Hunt,
1970
), and it is also involved in the development of the salivary
gland duct (Jones et al.,
1998
). Our results demonstrate that eyg, and its related
and adjacent gene twin of eyegone (toe), play a role in the genetic
subdivision of the thorax. The eyg/toe function is necessary
for scutum formation, and its ectopic activity in the scutellum transforms
this into a scutum-like tissue. eyg expression in the scutum is
regulated by a positive input of the Iro genes and pnr, and by the
repressor activity of the Hh and Dpp signalling pathways.
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MATERIALS AND METHODS |
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Drosophila strains
The following Drosophila strains were used in this work to
generate loss of function clones: Df(3L)iro
(Leyns et al., 1996);
eyg20MD1, Gal4-eyggv5 (generous gift
of Maria Dominguez); FRT80 iroDfM1/TM6
(Gomez-Skarmeta et al., 1996
);
FRT42ptc16/CyO, FRT40Atkva12/Cyo
(Nellen et al., 1996
);
FRT40A MadB1/Cyo
(Wiersdorff et al., 1996
);
FRT40Asmo3/Cyo (Strutt
and Mlodzik, 1997
); FRT2A eygSA2/TM6B; and
FRT2A mwh/TM3 Sb. The FLP/FRT technique
(Xu and Rubin, 1993
) was used
to generate loss of function clones. Larvae of the appropriate genotype were
heat shocked for 1 hour at 37°C, at different larval stages. The clones
were visualised in discs by either loss of GFP or of ß-galactosidase
expression.
For gain-of-function experiments, the following Gal4 lines were used:
ywf36a hsFLP122; abx FRT f+ FRT
Gal4-UAS-lacZ/Cyo (de Celis and Bray,
1997); ywflp122; act-FRT y+ FRT Gal4 UASGFP/SM5
Tb (Ito et al., 1997
);
ap-Gal4 (Rincon-Limas et al.,
2000
); 455-Gal4
(Martin-Blanco, 1998
);
ush-Gal4 (Calleja et al.,
2002
); 248-Gal4
(Sanchez et al., 1997
);
pnr-Gal4 (Heitzler et al.,
1996
); and 638-Gal4 (M. Calleja and G.M., unpublished).
The gain-of-function clones were generated by recombination at the FRT
sequences. The gain of Gal4 and lacZ activity can be
detected by ß-Gal staining. In the adult cuticle, the clones can be
scored because they are mutant for f36a and contain
y+ activity. The UAS-ara
(Diez del Corral et al., 1999
)
and UAS-tkvQD
(Hoodless et al., 1996
) lines
used have been described previously. UAS-eyg and UAS-toe
lines were generated by cloning the whole ORF into the pUAST vector, using the
following cloning sites: NotI/XhoI for eyg, and
EcoRI/XhoI for toe. The constructs were injected
into yw embryos and stable lines were selected by rescue of the
white phenotype. eyg cDNA was kindly provided by C. Desplan,
and the toe cDNA by the BDGP. For the apoptosis experiments we used
an UAS-P35 line (Hay et al.,
1994
).
The lacZ reporter lines used were: en-LacZ
(Simcox et al., 1991),
neu-LacZ (Flybase) and esg-lacZ
(Whiteley et al., 1992
).
P-element mutagenesis
Males homozygous for the pGal4 insertion (EM461) were crossed to females
carrying the hop transposase. Excisions of the pGal4 transposon were selected
by the loss of the w+ eye in the F1 progeny. Individual
revertants were crossed to TM3/TM6 flies and balanced. PCR analysis was
carried out with individual stocks with strong phenotypes over
Df(3L)iro. We used one primer located 2.5 kb downstream of the
P-element insertion site and two primers located upstream of the insertion
site, one 3 kb upstream and the other 11.5 kb upstream.
Downstream primer, 5'-CCGGTGGACTATGGCGCGAACGGACGCG-3';
Upstream 1, 5'-CGGCGTGGCCACCTTGGGCTTTGAGCC-3'; and
Upstream 2, 5'-CGGCGAGGGGAGTGGGGCCTGATGGG-3'.
Generation of a polyclonal anti-Eyg antibody
The complete eyg ORF was cloned in the pQE vectors (Qiagen) and
the recombinant protein purified by following the QIA express protocols. The
protein was injected into guinea pigs and the serum obtained was used as a
polyclonal antibody.
Inmunostaining of embryos and discs
Discs were dissected in PBS and fixed in 4% paraformaldehyde for 20 minutes
at room temperature. Discs were subsequently washed in PBS, blocked in
blocking buffer (PBS, 0.3% Triton, 1% BSA), and incubated overnight with the
primary antibody: anti-ß-Gal (1:2000; rabbit), anti-Eyg (1:200; guinea
pig) or anti-Ara (1:200; rat) diluted in blocking buffer at 4°C. Washes
were performed in blocking buffer, and the appropriate fluorescent secondary
antibody was added for 1 hour at room temperature. Following further washes in
blocking buffer, the discs were mounted in Vectashield. Anti-Ara antibody was
kindly by S. Campuzano, anti-ß-Gal (rabbit) and anti-caspase 3 were
purchased from Cappel and from Cell Signalling, respectively. Images were
taken in a laser MicroRadiance microscope (Bio-Rad) and subsequently processed
using Adobe Photoshop.
In situ hybridisation and antibody/in situ hybridisation-double labelling
were performed as described (Azpiazu and
Frasch, 1993), and embryos were mounted in Permount (Fisher
Scientific). Digoxigenin-labelled RNA probes were synthesised as described
(Tautz and Pfeifle, 1989
).
eyg-specific antisense RNA probe was synthesised from a 570 bp
SacI/KpnI fragment of the cDNA provided by C. Desplan. The
toe-specific antisense RNA probe was synthesised from a plasmid
provided by the BDGP and consists of an 475 bp EarI/XhoI
fragment.
Preparation of larval and adult cuticles
Adult flies were prepared by the standard methods for microscopic
inspection. Soft parts were digested with 10% KOH, washed with alcohol and
mounted in Euparal. Embryos were collected overnight and aged an additional 12
hours. First instar larvae were dechorionated in commercial bleach for 3
minutes and the vitelline membrane removed using heptano-methanol 1:1. Then,
after washing with methanol and 0.1% Triton X-100, larvae were mounted in
Hoyer's lactic acid (1:1) and allowed to clear at 65°C for at least 24
hours.
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RESULTS |
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By using plasmid rescue, we cloned sequences at the insertion point of the P-element (see Materials and Methods), and found that it was inserted 102 bp 5' of the transcription start site of eyg (FlyBase). The expression pattern found in EM461>UAS-lacZ embryos was similar to that of eyg (see below). Also, the imaginal expressions of EM461 and of eyg were largely coincident.
The analysis of the genomic region flanking the insertion point revealed the presence of another transcription unit, toe, and one predicted gene (CG32102; Fig. 1). toe is located 35.7 kb upstream from the P-element insertion, and CG32102 1.1 kb downstream from the insertion point. No cDNA has been reported for the latter gene. By contrast, toe has been previously cloned and shows high sequence identity with eyg (FlyBase). Their sequence conservation and location indicates that eyg and toe derive from a duplication of a primordial eyg-like gene, and raises the possibility that they may have redundant functions.
The expression domains of eyg and toe
We have analysed the expression of eyg and toe during
embryonic and imaginal disc development using the UAS-lacZ construct,
eyg and toe specific RNA probes, and an anti-Eyg polyclonal
antibody generated in our laboratory. The distribution of the eyg and
toe transcripts appears to be very similar during embryogenesis, and
also in the wing imaginal disc, although the expression levels of toe
are consistently lower than those of eyg. The ß-Gal distribution
in eyg-Gal4/UAS-lacZ embryos and imaginal discs matches that observed
for the eyg and toe transcripts, and that observed with the
anti-Eyg antibody. Thus, we shall use the anti-Eyg antibody as indicative of
the expression of both genes.
Our results on the embryonic expression of eyg/toe are in
agreement with those already reported for eyg using in situ
hybridisation (Jones et al.,
1998) (see also Fig.
3), and will not be considered further. We checked the possibility
that eyg might be expressed in the embryonic primordia of dorsal
discs by double-label experiments with escargot (esg)
(Whiteley et al., 1992
) and
found that none of the cells expressing eyg show esg
activity (data not shown). This result suggests that at the beginning of the
wing disc development eyg is not yet active.
eyg/toe is expressed in the eye, wing and haltere discs. The
expression and function of eyg in the eye disc has been reported
(Hazelett et al., 1998
);
expression in the wing disc is shown in
Fig. 2. The first sign of
activity is observed at the beginning of the third instar
(Fig. 2B), and, by that stage,
the Eyg product already is restricted to a part of the thoracic region of the
disc. In mature discs there are, in addition to the major domain in the
thorax, two small expression domains in the hinge
(Fig. 2B,D) and the pleura.
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The general conclusion from these experiments is that in the wing disc eyg/toe expression is activated approximately at the onset of the third larval period. Its expression is restricted from the beginning to a subgroup of thoracic cells that will form the anterior central portion of the mesothorax.
Loss- and gain-of-function studies of eyg and
toe
Phenotype of eyg mutations in the notum
We have generated a large number of revertants of the eyg-Gal4
line that lose the w+ gene. Of these, the
eygSA1 and eygSA2
revertants fail to complement and their trans combination shows a clear notum
phenotype, lacking most of the bristles. They also fail to complement
eyg20MD1, a P-lacZ insertion located 3' of
the eyg gene. The eygSA2 and
eyg20MD1 alleles show a stronger phenotype in their trans
combinations, and over Df(3L)iro, a deletion of eyg, toe and
several other genes (see Materials and Methods). eygSA2 is
a 9 kb deletion including the entire eyg transcription unit, but not
that of toe. eygSA1 is a 3.8 kb deletion that removes part
of the eyg transcription unit. Most of the phenotypic analysis has
been carried out using the eygSA2 allele and the
Df(3L)iro. There are no individual mutants for the toe
gene.
The phenotype of eygSA2/Df(3L)iro is shown in Fig. 3A. The notum is much reduced owing to the lack of practically the entire eyg/toe domain. The majority of the microchaetes of the central region and the dorsocentral bristles are missing, but the scutellar and lateral bristles are present. The zones not affected by the mutations are those that in wild-type flies do not possess eyg/toe activity.
As most of the normal eyg domain is lacking in
eygSA2/Df(3L)iro flies, there was the possibility that the
loss of eyg function may cause apoptosis in cells normally expressing
eyg. It has been reported that eyg mutants produce apoptosis
in the eye cells anterior to the morphogenetic furrow
(Jang et al., 2003). However,
in our experiments, we do not find apoptosis in the mutant wing discs and
there is no detectable caspase activity in the notum region (using an
anti-caspase antibody; data not shown). Moreover, we carried out an experiment
designed to assay the contribution of apoptosis to the eyg-mutant
phenotype. The mutation eyggv5 is a Gal4 insertion at
eyg with a strong phenotype (Fig.
3H), which can be used to drive the activity of the baculovirus
gene P35, a general inhibitor of apoptosis
(Hay et al., 1994
). Flies of
the genotype eyggv5/Df(3L)iro; UAS-P35 show the same
phenotype as their siblings eyggv5/Df(3L)iro, indicating
that apoptosis is not a major factor contributing to the eyg syndrome
in the notum.
We have also induced eyg-mutant clones cells in the notum. As expected, these clones behaved normally outside the eyg domain, but those inside the domain show loss of bristles and abnormal patterning, which is often associated with alterations in the normal polarity of surrounding bristles and trichomes. In the disc they adopt a round shape, suggesting that they tend to sort out from surrounding eyg-expressing cells. These results indicate that the activity of eyg is connected with the acquisition of specific cell affinity properties.
The interpretation of the phenotype of eyg mutations may be complicated by the possibility that although the eygSA2 is a complete loss-of-eyg transcription, there could be some rescue by toe, which is expressed in the same domain. However, we note that, in eyg-mutant flies, most or all the eyg domain is lost (Fig. 3A,H), suggesting that if there is rescue by toe it has to be weak. The ability of the Toe product to rescue the eyg-mutant phenotype was tested in an experiment in which we used the eyggv5 Gal4 line to drive the Toe product. As shown in Fig. 3H,I, flies of the genotype eyggv5 > UAS-toe show partial rescue of the eyggv5 phenotype in the notum. This experiment indicates that the Toe product has a developmental potential similar to that of Eyg (see also below and Fig. 4). However, toe transcript levels appear normal in eygSA2 mutants (Fig. 3G), and still are not able to rescue the strong phenotype of eygSA2, suggesting that the normal toe levels are not sufficient to substitute for the loss of eyg activity.
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Altogether, the preceding results indicate that normal eyg function is required for the appearance of the anterior central part of the notum. There is little, if any, rescue of the eyg-mutant phenotype by toe, in spite of the fact that it is expressed in the same domain. The results also demonstrate that toe is not regulated by eyg, as the loss of eyg activity does not affect toe expression.
Misexpression of eyg and toe
To study the effects of ectopic eyg and toe expression by
the Brand and Perrimon method (Brand and
Perrimon, 1993), we generated UAS-eyg and
UAS-toe transformants, which then were crossed to various Gal4 lines.
We also used the flip-out method (Chou and
Perrimon, 1992
) to induce marked clones of eyg-expressing
cells.
The effect of misexpressing eyg and toe in the notum was analysed using the 248-Gal4, pnr-Gal4, 638-Gal4, ap-Gal4, 455-Gal4 and ush-Gal4 driver lines. The combinations of the Gal4 lines with UAS-eyg, and with UAS-toe, give essentially the same results (Fig. 4A-C), indicating that the two gene products have a similar, or the same, developmental capacity.
In the combinations with 248-Gal4, 455-Gal4, 638-Gal4, ush-Gal4 and pnr-Gal4 the scutellum is modified and develops a scutum-like pattern, as indicated by shape alterations and the appearance of microchaetes (Fig. 4A-E). We also observe that there is an abrupt change in polarity of the bristles and trichomae close to the border of the transformation, suggesting that eyg and toe might have a role in defining polarity of the epidermal elements.
In contrast to the preceding combinations, in ap-Gal4/UAS-eyg
flies, the scutellum is absent but there is no indication of scutellum to
scutum transformation (Fig.
4F). As ap is expressed in all notal cells after the
second larval period (Diaz-Benjumea and
Cohen, 1993; Rincon-Limas et
al., 2000
) and precedes eyg expression, we believe that
inducing the activity of eyg early in notum development programmes
the whole notum to develop as the eyg/toe domain, thus
preventing the formation of scutellum (see Discussion).
The transformations observed in clones of eyg-expressing cells (data not shown) fit well with those described with the Gal4 lines. In the normal eyg territory these clones develop normally, indicating that increased levels of eyg activity do not appear to have a major effect. In the scutellum and lateral region they tend to sort out from surrounding tissue and also exhibit a change of fate; this is especially clear in the scutellum, where they develop scutum-like tissue.
These results suggest that eyg and toe are involved in the determination of the specific development of the anterior central region of the notum. This function is also reflected by the acquisition of specific cell affinities, which may have a role in the partitioning of the thorax (see Discussion). eyg- expressing clones in the disc are of normal shape in the eyg domain but have a round shape in the scutellum region, again indicating that eyg affects cell affinities.
eyg requires Iro gene activity to specify notum
patterns
One significant result of the misexpression experiments is that whereas
ectopic eyg or toe expression in the thorax induces
scutum-like tissue, their activity in the wing does not produce a comparable
transformation. The expression of eyg, or of toe, alone
produces defects in growth and differentiation in the wing, but there is no
clear indication of transformation towards notum
(Fig. 5B). This behaviour is
clearly illustrated by the 638-Gal4 line which drives expression in
the posterior notum as well as in the wing pouch
(Fig. 5A). In 638-Gal4 >
UAS-eyg flies the scutellum, but not the wing, is transformed towards
scutum. Only in a small region in the centre of the wing is there is some
tissue resembling notum (Fig.
5B).
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Flies of genotype 638-Gal4 > UAS-ara exhibit normal notum
development and in addition the wing also becomes transformed towards notum
(Fig. 5C). Thus the Ara product
has the capacity to induce notum development, as expected from previous
studies (Diez del Corral et al.,
1999). In the favourable cases, it is possible to discriminate the
presence of scutellum structures in the ectopic notum
(Fig. 5C). In 638-Gal4 >
UAS-ara UAS-eyg flies, ara and eyg are co-expressed in
the same cells of the wing. In these flies, the ectopic notum produced in
place of the wing displays, additionally, a scutellum to scutum transformation
(Fig. 5D). This result strongly
suggests that ara (and probably cau) is providing the
necessary `thoracic' context for the normal function of
eyg/toe. We believe that the notum-like tissue observed in
the centre of the wing of 638-Gal4 > UAS-eyg flies
(Fig. 5B) is caused by the Iro
gene activity normally present in that region
(Gomez-Skarmeta et al.,
1996
).
Altogether the results obtained about the developmental consequences of eliminating or misexpressing eyg and toe indicate that these genes have a role in the subdivision of the notum into an anterior-central and a posterior-lateral region. To achieve this function they require the contribution of the Iro genes.
Regulation of eyg expression in the notum
The eyg/toe expression domain in the thorax covers only
part of the notum; as illustrated in Fig.
2, eyg/toe-expressing cells in the mature disc are
restricted to the anterior and central region of the disc. The misexpression
experiments show that this spatial restriction is important for the normal
patterning of the thorax. Thus, the factors controlling eyg/toe
expression are significant components of the pattern formation process of the
thorax.
The Iro genes and pnr upregulate eygexpression
We first checked the regulatory role of the Iro genes, for although in
mature discs eyg and the Iro genes are not co-extensive
(Fig. 2D), there is evidence
that in early disc development Iro gene expression covers much or all the
mesothorax (Diez del Corral et al.,
1999). Thus the eyg domain would be a subset of the initial Iro
gene domain, which suggests that the Iro genes might function as early
activators of eyg. Moreover, cells lacking Iro gene activity are not
able to differentiate thoracic structures
(Diez del Corral et al.,
1999
), suggesting that they also lack eyg activity.
We find that, in absence of Iro gene activity, eyg expression is
lost: clones of Iro gene mutant cells induced in the first instar abolish
eyg expression even in regions in which the Iro genes are not active
in mature discs (Fig. 6A-C). An
intriguing feature of these clones is that their effect on eyg
extends to the zone outside the clone. Similar non-autonomous effect of the
Iro genes have been reported (Diez del
Corral et al., 1999).
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The hh and Dpp pathways repress eyg activity
Another important aspect of eyg regulation is how its expression
is inhibited in the scutellum and lateral notum. These regions are close to
the AP compartment border, suggesting that the Hh and Dpp pathways might have
a repressing role. There is evidence
(Mullor et al., 1997) that the
scutellum and the zone close to the AP border is patterned by Hh. This region
is also expected to contain high levels of Dpp.
The role of the Hh pathway was tested by examining eyg expression in clones of cells that either are defective in [by eliminating the activity of the smoothened (smo) transducer] or express high levels of hh activity (by removing ptc activity or misexpressing hh). As illustrated in Fig. 7A-C, smo- clones located in the proximity of the AP border gain eyg activity. This gain of eyg activity is also reflected in the cuticular differentiation of these clones, which form scutum-like structures (Fig. 7D). On the contrary, ptc- clones localised in the eyg domain show loss of eyg expression (Fig. 7E-G). Similarly, ectopic expression of hh in the eyg domain results in loss of eyg expression (data not shown).
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DISCUSSION |
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As are the other segmental structures of Drosophila, the
mesothorax is subdivided into anterior (notum) and posterior (postnotum)
compartments by the activity of the en gene. Subsequently, these
compartments are further subdivided
(Calleja et al., 2000) into
medial and lateral regions by the activities of the pnr and Iro
genes. These regions are not defined by restricted lineages, but are kept
developmentally segregated by the differential affinities of the pnr-
and Iro gene-expressing cells (reviewed by
Mann and Morata, 2000
;
Calleja et al., 2000
). We now
find that the notum, but not the postnotum, is further subdivided by the
activity of eyg/toe. Its role in this subdivision is clearly
demonstrated both by the experiments in which its function is eliminated and
when it is ectopically expressed. The lack of eyg results in flies in
which the central anterior region, corresponding to the
eyg/toe notum domain, is missing
(Fig. 3A). Conversely, ectopic
expression of either eyg or toe in the scutellum transforms
it towards scutum (Fig. 4). The
conclusion is that eyg/toe is involved in a new step in the
subdivision of notum, which is superimposed over the previous one established
by the Iro genes and pnr. The result is the appearance of four
distinct regions, according to the active or inactive state of
eyg/toe in the pnr and the Iro gene domains. The specific
activation of eyg/toe in the notum, the anterior mesothoracic
compartment, but not in the postnotum, the posterior mesothoracic compartment,
appears to be an important factor in the generation of diversity in the
thorax.
A significant functional feature of eyg/toe is that it is unable
to induce notum structures by itself, but requires co-expression of its
activator the Iro gene (Fig.
5), and probably pnr. For example, whereas ectopic
eyg/toe activity induces scutum-like structures in the scutellum
(which is also part of the notum and which expresses pnr), it fails
to do so in most of the wing. Interestingly, it only induces notal structures
in the middle of the wing (Fig.
5B), precisely the place where there is Iro gene activity in
normal development (Gomez-Skarmeta et al.,
1996). This mode of action is unlike that of selector or
selector-like genes, such as the Hox genes, en, Dll, pnr or the Iro
genes (reviewed by Mann and Morata,
2000
), which are able to induce, out of context, the formation of
the patterns they specify. This indicates that eyg/toe is not of the
same rank, but that it is developmentally downstream of the Iro genes and
pnr, and appears to mediate their `thoracic' function. The
restriction of eyg/toe activity to the thorax, unlike the Iro genes
and pnr, which are also expressed in the abdomen, is fully consistent
with this role. We note that eyg/toe is also expressed in a similar
domain in the metathorax, suggesting that it may perform a parallel role in
this segment.
The elaboration of the eyg/toe expression domain in the
notum
The eyg/toe expression domain occupies the larger part of
the notum, extending from the anterior border to the suture between the scutum
and the scutellum. This domain coincides with the region affected in the
eyg mutations, and is consistent with the gain-of-function
experiments. Thus the eyg/toe expression domain corresponds
to the zone where eyg/toe function is required. As it is only a part
of the notum, a question of interest is to find out how eyg/toe
expression is restricted to this zone. This restriction is necessary for the
appearance of distinct anterior-central and posterior-lateral subdomains, for
if eyg/toe are expressed uniformly, as in ap-Gal4 >
UAS-eyg flies (Fig. 4F), the entire notum develops as the anterior-central domain.
Our experiments indicate that the localised expression of eyg/toe is achieved by the activity of two antagonistic factors: the promoting activity induced by the Iro and pnr genes, and the repressing activities exerted by the Hh and the Dpp pathways. The latter are probably mediated by Hh and Dpp target genes that are yet to be identified.
Both Iro genes and pnr act as activators of eyg/toe
expression. In Iro gene-mutant clones eyg is abolished, and ectopic
Iro gene activity results in ectopic eyg expression
(Fig. 6). Although
pnr- clones do not lose eyg activity, the
probable explanation is that they show Iro gene activity
(Calleja et al., 2000), which
upregulates eyg. However, ectopic pnr expression induces
eyg activity (Fig. 6). Because the Iro gene and pnr expression domains cover the entire
notum, in the absence of any other regulation they would induce eyg
activity in the whole structure.
The elimination of eyg activity from the scutellum and lateral
notum is caused by the Hh and Dpp pathways. Because the AP compartment border
is displaced posteriorly in the notum, these two pathways are active at high
levels in the posterior region of the mesothorax. Assuming that the two
signals behave as in the wing (Lecuit et
al., 1996; Nellen et al.,
1996
), Hh activity will be higher in the region close to the AP
border, whereas the effect of Dpp will extend further anteriorly. Thus the
repressive role of Hh will be greater in the proximity of the AP border and
that of Dpp will be greater in more anterior positions. This is precisely what
our results indicate. In the region close to the AP border the high Hh levels
alone are sufficient to repress eyg
(Fig. 8C). However. in more
anterior positions, close to the border of the eyg domain, Hh levels
are lower and, although Hh is still necessary, it is not sufficient to repress
eyg. Here there is an additional requirement for Dpp activity
(Fig. 8A-F).
Thus, the part of the notum that does not express eyg can be subdivided into two distinct zones according to the mode of eyg regulation: a region close to the AP border that requires only Hh, and a more anterior region that requires both Hh and Dpp. In the posterior compartment, the repression of eyg has to be achieved by a different mechanism because neither the inactivation of the Hh pathway in smo- clones, nor the inactivation of the Dpp pathway (Fig. 8D-F), induces ectopic eyg activity. A probable possibility is that en itself may act as repressor.
The result of the antagonistic activities of the Iro genes and pnr on the one hand, and of the Hh and Dpp signalling pathways on the other, subdivides the notum into an eyg/toe expressing domain and a non-expressing domain. The localised expression of eyg/toe contributes to the morphological diversity of the thorax, as it distinguishes between an anterior-central region and a posterior-lateral one. It provides another example of a genetic subdivision of the body that is not based on lineage. It also provides an example of a patterning gene acting downstream of the combinatorial code of selector and selector-like genes. Its mode of action supports a model in which the genetic specification of complex patterns, such as the notum, is achieved by a stepwise process involving the activation of a cascade of regulatory genes.
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ACKNOWLEDGMENTS |
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REFERENCES |
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---|
Azpiazu, N. and Frasch, M. (1993). tinman and bagpipe: two homeo box genes that determine cell fates in the dorsal mesoderm of Drosophila. Genes Dev. 7,1325 -1340.[Abstract]
Basler, K. and Struhl, G. (1994). Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368,208 -214.[CrossRef][Medline]
Brand, A. H. and Perrimon, N. (1993). Targeted
gene expression as a means of altering cell fates and generating dominant
phenotypes. Development
118,401
-415.
Calleja, M., Moreno, E., Pelaz, S. and Morata, G.
(1996). Visualization of gene expression in living adult
Drosophila. Science
274,252
-255.
Calleja, M., Herranz, H., Estella, C., Casal, J., Lawrence, P.,
Simpson, P. and Morata, G. (2000). Generation of medial and
lateral dorsal body domains by the pannier gene of Drosophila.
Development 127,3971
-3980.
Calleja, M., Renaud, O., Usui, K., Pistillo, D., Morata, G. and Simpson, P. (2002). How to pattern an epithelium: lessons from achaete-scute regulation on the notum of Drosophila. Gene 292,1 -12.[CrossRef][Medline]
Chou, T.-B. and Perrimon, N. (1992). Use of a
yeast site specific recombinase to produce female germline chimeras in
Drosophila. Genetics
131,643
-653.
de Celis, J. F. and Bray, S. (1997). Feed-back
mechanisms affecting Notch activation at the dorsoventral boundary in the
Drosophila wing. Development
124,3241
-3251.
Diaz-Benjumea, F. J. and Cohen, S. M. (1993). Interaction between dorsal and ventral cells in the imaginal disc directs wing development in Drosophila. Cell 75,741 -752.[Medline]
Diez del Corral, R., Aroca, P., Gomez-Skarmeta, J. L.,
Cavodeassi, F. and Modolell, J. (1999). The Iroquois
homeodomain proteins are required to specify body wall identity in Drosophila.
Genes Dev. 13,1754
-1761.
Gomez-Skarmeta, J. L., Diez del Corral, R., de la Calle-Mustienes, E., Ferres-Marco, D. and Modolell, J. (1996). araucan and caupolican, two members of the novel iroquois complex, encode homeoproteins that control proneural and vein-forming genes. Cell 85, 95-105.[Medline]
Hay, B. A., Wolff, T. and Rubin, G. (1994).
Expression of baculovirus P35 prevents cell death in Drosophila
Development 120,2121
-2129.
Hazelett, D. J., Bourouis, M., Walldorf, U. and Treisman, J.
E. (1998). decapentaplegic and wingless are
regulated by eyes absent and eyegone and interact to direct
the pattern of retinal differentiation in the eye disc.
Development 125,3741
-3751.
Heitzler, P., Haenlin, M., Ramain, P., Calleja, M. and Simpson,
P. (1996). A genetic analysis of pannier, a gene necessary
for viability of dorsal tissues and bristle positioning in Drosophila.
Genetics 143,1271
-1286.
Herranz, H. and Morata, G. (2001). The
functions of pannier during Drosophila embryogenesis.
Development 128,4837
-4846.
Hoodless, P. A., Haerry, T., Abdollah, S., Stapleton, M., O'Connor, M. B., Attisano, L. and Wrana, J. L. (1996). MADR1, a MAD-related protein that functions in BMP2 signaling pathways. Cell 85,489 -500.[Medline]
Hunt, D. M. (1970). Lethal interactions of the eye-gone and eyeless mutants in Drosophila melanogaster. Genet. Res. 15,29 -34.[Medline]
Ito, K., Awano, W., Suzuki, K., Hiromi, Y. and Yamamoto, D.
(1997). The Drosophila mushroom body is a quadruple structure of
clonal units each of which contains a virtually identical set of neurones and
glial cells. Development
124,761
-771.
Jang, C. C., Chao, J. L., Jones, N., Yao, L. C., Bessarab, D.
A., Kuo, Y. M., Jun, S., Desplan, C., Beckendorf, S. K. and Sun, Y.
H. (2003). Two Pax genes, eyegone and
eyeless, act cooperatively in promoting Drosophila eye
development. Development
130,2939
-2951.
Jones, N. A., Kuo, Y. M., Sun, Y. H. and Beckendorf, S. K.
(1998). The Drosophila Pax gene eye gone is
required for embryonic salivary duct development.
Development 125,4163
-4174.
Jun, S. and Desplan, C. (1996). Cooperative
interactions between paired domain and homeodomain.
Development 122,2639
-2650.
Lawrence, P. and Morata, G. (1977). The early development of mesothoracic compartments in Drosophila. An analysis of cell lineage and fate mapping and an assesment of methods. Dev. Biol. 56,40 -51.[Medline]
Lawrence, P. and Morata, G. (1994). Homeobox genes: their function in Drosophila segmentation and pattern formation. Cell 78,181 -189.[Medline]
Lawrence, P. and Struhl, G. (1996). Morphogens, compartments and pattern: lessons from Drosophila? Cell 85,951 -961.[Medline]
Lecuit, T., Brook, W. J., Ng, M., Calleja, M., Sun, H. and Cohen, S. M. (1996). Two distinct mechanisms for long-range patterning by Decapentaplegic in the Drosophila wing. Nature 381,387 -393.[CrossRef][Medline]
Leyns, L., Gomez-Skarmeta, J. L. and Dambly-Chaudiere, C. (1996). iroquois: a prepattern gene that controls the formation of bristles on the thorax of Drosophila. Mech. Dev. 59, 63-72.[CrossRef][Medline]
Mann, R. and Morata, G. (2000). The developmental and molecular biology of genes the subdivide the body of Drosophila. Annu. Rev. Cell Dev. Biol. 16,243 -271.[CrossRef][Medline]
Martin-Blanco, E. (1998). Regulatory control of signal transduction during morphogenesis in Drosophila. Int. J. Dev. Biol. 42,363 -368.[Medline]
Morata, G. and Lawrence, P. A. (1975). Control of compartment development by the engrailed gene in Drosophila. Nature 255,614 -617.[Medline]
Mullor, J. L., Calleja, M., Capdevila, J. and Guerrero, I.
(1997). Hedgehog activity, independent of decapentaplegig,
participates in wing disc patterning. Development
124,1227
-1237.
Nellen, D., Burke, R., Struhl, G. and Basler, K. (1996). Direct and long-range action of a DPP morphogen gradient. Cell 85,357 -368.[Medline]
Newfeld, S. J., Chartoff, E., Graff, J. M., Melton, D. A. and
Gelbart, W. M. (1996). Mothers against dpp encodes a
conserved cytoplasmic protein required in DPP/TGF-ß responsive cells.
Development 122,2099
-2108.
Rincon-Limas, D. E., Lu, C. H., Canal, I. and Botas, J.
(2000). The level of DLDB/CHIP controls the activity of the LIM
homeodomain protein apterous: evidence for a functional tetramer complex in
vivo. EMBO J. 19,2602
-2614.
Sanchez, L., Casares, F., Gorfinkiel, N. and Guerrero, I. (1997). The genital disc of Drosophila melanogaster. Dev. Genes Evol. 207,219 -241.
Simcox, A. A., Hersperger, E., Shearn, A., Whittle, J. R. S. and Cohen, S. M. (1991). Establishment of imaginal disc and histoblast nests in Drosophila. Mech. Dev. 34, 11-20.[CrossRef][Medline]
Strutt, D. I. and Mlodzik, M. (1997). Hedgehog
is an indirect regulator of morphogenetic furrow progression in the Drosophila
eye disc. Development
124,3233
-3240.
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85.[Medline]
Whiteley, M., Noguchi, P. D., Sensabaugh, S. M., Odenwald, W. F. and Kassis, J. A. (1992). The Drosophila gene escargot encodes a zinc finger motif found in snail-related genes. Mech. Dev. 36,117 -127.[CrossRef][Medline]
Wiersdorff, V., Lecuit, T., Cohen, S. M. and Mlodzik, M.
(1996). Mad acts downstream of Dpp receptors, revealing a
differential requirement for dpp signaling in initiation and propagation of
morphogenesis in the Drosophila eye. Development
122,2153
-2162.
Xu, T. and Rubin, G. M. (1993). Analysis of
genetic mosaics in developing and adult Drosophila tissues.
Development 117,1223
-1237.