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First published online 20 August 2008
doi: 10.1242/dev.025809
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Centro de Biología Molecular Severo Ochoa (C.S.I.C.-U.A.M.), Nicolás Cabrera 1, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
* Author for correspondence (e-mail: esherrero{at}cbm.uam.es)
Accepted 31 July 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Hox, Ultrabithorax, Polycomb, Autoregulation, engrailed
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Duboule, 2007). They are
expressed in defined domains along this axis and their mis-expression
frequently causes gross alterations in the body plan. Therefore, Hox gene
expression must be tightly regulated throughout development (reviewed by
Carroll et al., 2001).
In Drosophila, the expression domains of Hox genes are set in the
early embryo by the activity of gap genes
(White and Lehmann, 1986;
Harding and Levine, 1988;
Irish et al., 1988;
Casares and Sánchez-Herrero,
1996). After this initial regulation, Hox genes expression domains
are maintained by two groups of genes: the Polycomb (Pc)
group, which code for proteins that maintain the repression of Hox genes (and
other genes); and the trithorax (trx) group, coding for
proteins that maintain Hox transcription by preventing Pc silencing
(reviewed by Schwartz and Pirrotta,
2007). Pc-group complexes bind to DNA in specific regions
called Polycomb response elements (PREs) (reviewed by
Müller and Kassis, 2006;
Ringrose and Paro, 2007). Hox
expression is also regulated by the Hox genes themselves: those expressed more
posteriorly along the A/P axis downregulate the expression of those
transcribed more anteriorly (Hafen et al.,
1984; Struhl and White,
1985). Finally, some Hox genes control their own expression. For
example, Deformed maintains its own transcription in cells of the
epidermis and central nervous system
(Kuziora and McGinnis, 1988;
Lou et al., 1995). The
opposite effect, negative regulation by its own product, has been described
for the Ultrabithorax (Ubx) gene
(Irvine et al., 1993).
Ubx expression in the embryonic epidermis extends from parasegment
(PS) 5 to PS12. In the larval thorax, Ubx is expressed in imaginal
discs of the third thoracic segment (haltere disc and third leg disc) and in
the posterior compartment of the second leg disc
(Beachy et al., 1985;
White and Wilcox, 1985). In
this region, Ubx mutations transform the third leg into the second
one, and the haltere and metanotum (proximal part of the dorsal metathorax)
into wing and mesonotum (corresponding region of the mesothorax), respectively
(Lewis, 1963).
Ubx regulates negatively its own expression in the embryonic
epidermis and imaginal discs (Irvine et
al., 1993; Casares et al.,
1997). Thus, increasing the amount of Ubx protein reduces
Ubx transcription, and the opposite effect is seen with mutations
that reduce its expression (Irvine et al.,
1993). Changes in Ubx levels also modify the adult
phenotype, as increasing Ubx dose reduces haltere size
(Smolik-Utlaut, 1990;
Irvine et al., 1993);
conversely, heat-shock-induced Ubx expression occasionally brings
about a very slight transformation of haltere into wing
(Irvine et al., 1993). This
transformation is particularly intriguing, as adding more Ubx seems
to reduce Ubx activity.
We have studied this surprising result and have found that the increased expression of Ubx produces a strong Ubx mutant phenotype, but only if the high Ubx protein levels are transitorily present in the imaginal disc cells. The transient high Ubx expression causes a Pc-group-dependent permanent inactivation of Ubx, probably owing to the repression of Ubx endogenous transcription. A similar effect is also observed in engrailed (en), a gene required to specify the development of posterior compartments. The mechanism we have uncovered, therefore, seems to be at work in different genes showing negative auto-regulation in Drosophila development.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) was used with the following drivers: MS372-Gal4 (a
gift from F. Jiménez), Ubx-Gal4SS.2 (A.
Sánchez and E.S.-H.), C-765-Gal4
(Guillén et al., 1995),
sd-Gal4 (M.C. and G. Morata, unpublished), Ubx-Gal4M1,
Ubx-Gal4M3, dpp-Gal4, en-Gal4, ptc-Gal4,
ap-Gal4, hh-Gal4 (FlyBase;
http://flybase.bio.indiana.edu)
and with the following UAS constructs: UAS-Ubx
(Castelli-Gair et al., 1994;
Michelson, 1994),
UAS-Ubx-HA (Ronshaugen et al.,
2002), UAS-dsRNA>Ubx
(Monier et al., 2005),
UAS-abd-A (Michelson,
1994), UAS-Abd-B (m)
(Castelli-Gair et al., 1994),
UAS-en (Guillén et al.,
1995), UAS-UbxUbdA,
UAS-UbxHX (Merabet et
al., 2007), UAS-UbxAf (from Artemia franciscana)
(Ronshaugen et al., 2002) and
UAS-Pcl-RNAi (Vienna Drosophila RNAi Center). The
Pc3 and trxE2mutations, the
Df109 deletion, which eliminates Ubx, and the UAS-GFP
reporter construct are described in FlyBase. P-lacZ lines inserted in
the Ubx (Ubxlac1)
(Casares et al., 1997),
abd-A (HC7JA1), Abd-B (HCJ199)
(Bender and Hudson, 2000) and
en (ryxho25) (Hama et
al., 1990) were used as reporters of expression of the
corresponding genes.
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). The
Gal80ts/Gal4 system (McGuire et
al., 2003) was used to control the time of expression of the
exogenous Ubx. Clones were marked by the absence of GFP expression
and induced according to the following protocol: larvae at 48-72 hours of
development, raised at 25°C, were heat-shocked at 37°C for 50 minutes
to induce recombination between the FRT sequences, transferred to 29°C for
2 days (to allow Gal4 activity) and then transferred to 17°C to inactivate
the Gal4 protein (McGuire et al.,
2003). After several days at 17°C, the discs were fixed and
scored for clones. In this and similar experiments with several temperature
changes, the time of pupation is delayed.
Clones eliminating Pc function in a background in which
Ubx permanent repression was established were induced according to
this procedure: 24-48 hour larvae of the genotype
sd-Gal4/hs-flp;
UAS-Ubx/tub-Gal80ts; Ubi-GFP
FRT2A/hs-CD2 ri PcXT109 FRT2A, grown at 25°C,
were transferred to 29°C for 24 hours, heat-shocked for 1 hour at
37°C, grown for 1 day at 29°C and transferred to 17°C for 2 days
before fixation. The hs-CD2 ri FRT2A
PcXT109 chromosome is described by Beuchle et al.
(Beuchle et al., 2001).
Immunostaining
Imaginal discs were stained according to standard procedures. The
antibodies used are mouse and rabbit anti-β-galactosidase (Cappel), mouse
Mab4D9 anti-En (Patel et al.,
1989), rat anti-haemagglutinin (Roche) and mouse anti-Ubx
(White and Wilcox, 1984).
Secondary antibodies are coupled to Red-X, Texas Red, FITC and Cy5
fluorochromes (Jackson ImmunoResearch).
In situ hybridization
Haltere discs were hybridized with a Ubx cDNA probe according to
standard protocols (Wolff,
2000).
Adult cuticle analysis
Flies were kept in a mixture of ethanol: glycerol (3:1), cooked in 10% KOH
at 60°C for 10 minutes, dissected, washed with water, dehydrated with
ethanol and mounted in Euparal for inspection under a compound microscope.
X-gal staining and inverse PCR
X-gal staining was carried out as previously described
(Wolff, 2000). The P-Gal4 line
Ubx-Gal4SS.2 was localized by inverse PCR
(http://www.fruitfly.org/about/methods/inverse.pcr.html).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). This is
easily observed when expressing Ubx with the Gal4/UAS system
(Brand and Perrimon, 1993) in
haltere discs of larvae carrying a Ubx-lacZ reporter insertion,
Ubxlac1 (Casares et
al., 1997). In these discs, there is strong reduction of
β-galactosidase expression, indicating repression of the endogenous
Ubx gene by the exogenous Ubx protein
(Fig. 1B, compare with the
lacZ expression in Ubxlac1 discs in
Fig. 1A). Flies with increased
Ubx expression present, in some GAL4/UAS combinations, a partial
transformation of haltere into wing (Fig.
1D,E, compare with the wild type in
Fig. 1C). A weak effect on
haltere development has been described after expressing Ubx under heat-shock
control (Irvine et al., 1993)
but the transformations we observe are much stronger. Other regions of the
third thoracic segment (metanotum and third leg) are also transformed into the
second thoracic one (see below). By contrast, Ubx expression driven
by other Gal4 lines do not transform the halteres into wings but reduce their
size. This is similar to what has been previously described
(Smolik-Utlaut, 1990;
Irvine et al., 1993), and
suggests that there may be some peculiarity in the Gal4 drivers that produce
Ubx transformations when expressing Ubx. These transformations are paradoxical, as providing an excess of Ubx protein results in a phenotype similar to that produced when eliminating the Ubx product. We wondered whether there could be, in addition to the repression of the endogenous Ubx gene, a dominant-negative effect caused by an excess of Ubx protein or whether, by contrast, total Ubx protein levels were reduced. To check this, we stained third instar haltere discs of the MS372-Gal4/UAS-Ubx combination, which produces Ubx transformations (Fig. 1E), with an anti-Ubx antibody. As shown in Fig. 1F, there are patches of cells that lack Ubx expression. Therefore, the Ubx phenotype is due to the absence of the Ubx protein.
High transient expression of exogenous Ubx accounts for the permanent absence of Ubx protein
A question posed by the previous experiments is why only some Gal4 lines
elicit Ubx mutant transformations. It could be that these lines are
repressed by Ubx. We studied whether this is the case with the
MS372-Gal4 line, which drives expression in the haltere disc
(Fig. 2A). To distinguish the
contribution of endogenous and exogenous Ubx proteins to the Ubx
pattern, we monitored the endogenous Ubx gene (Endo-Ubx)
with the Ubxlac1 insertion and the exogenous one
(Exo-Ubx) by using an antibody against haemagglutinin (HA) in larvae
expressing a Ubx protein tagged with this epitope (Ubx-HA)
(Ronshaugen et al., 2002).
Several conclusions can be drawn when analyzing UAS-GFP/+;
MS372-Gal4/UAS-Ubx Ubxlac1
(Fig. 2A-A''') and
UAS-Ubx-HA/+; MS372-Gal4 UAS-GFP/Ubxlac1
(Fig. 2B-B'') haltere
discs: first, there are cells expressing both Ubx and GFP
(Fig. 2A,A',A'''),
as in the haltere discs of MS372-Gal4 UAS-GFP larvae, arguing that an
increase in Ubx levels does not repress the driver. Second, as
expected, repression of the Endo-Ubx gene coincides with absence of
the Ubx protein (Fig.
2A',A''). Third, the Exo-Ubx distribution
follows that of GFP (Fig. 2B),
but not all the cells expressing Exo-Ubx repress Endo-Ubx
(β-galactosidase signal) (Fig.
2B',B''), perhaps because of the perdurance of the
β-galactosidase protein. Finally, and significantly, some cells lack GFP,
Endo-Ubx and Exo-Ubx
(Fig. 2A'',B''',
insets). We hypothesize that these cells may derive from cells that expressed
the driver only transiently, so that Exo-Ubx and GFP are no longer
present, and in which Endo-Ubx transcription (previously repressed by
Exo-Ubx) has not started again.
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). In
ptc-Gal4 UAS-GFP/+;
UAS-Ubx/tub-Gal80ts or
tub-Gal80ts/+; dpp-Gal4 UAS-GFP/UAS-Ubx
haltere discs, when larvae were transferred from 17°C, the permissive
temperature of the Gal80ts protein, to 29°C, the restrictive
one, and back again to 17°C, Ubx is absent in patches of cells in
the anterior compartment, which exceptionally occupy a large domain
(Fig. 2C and data not shown).
It is known that the dpp expression domain is wider in early wing
discs than in late ones, where its transcription is confined to cells abutting
the anteroposterior boundary (Weigmann and
Cohen, 1999). Assuming similar changes occur in the haltere disc,
the transient Ubx expression driven by dpp-Gal4 (or
ptc-Gal4) away from this boundary in early larvae may account for the
absence of Ubx protein observed at late stages. Consistently, we see
transformations of haltere to wing in dpp-Gal4/UAS-Ubx flies
(not shown).
We have supposed that the transitory high expression of Ubx may
repress its own transcription permanently. To check this idea, and to avoid
complications using Gal4 lines with variable expression throughout
development, we decided to use the ap-Gal4 line, which drives
constant expression in the dorsal compartment of wing and haltere discs
(Calleja et al., 1996)
(Fig. 3A), and to temporally
restrict its activity with the Gal4/Gal80ts system. First, we
studied Endo-Ubx expression when there is a continuous supply of
Exo-Ubx. The dorsal compartment of ap-Gal4 UAS-GFP/+;
UAS-Ubx Ubxlac1/+ haltere disc shows high
Ubx levels (Fig.
3A') and complete repression of Endo-Ubx
(Fig. 3A''). We next
restricted the time when Exo-Ubx is synthesized. ap-Gal4
UAS-GFP/+; UAS-Ubx
Ubxlac1/tub-Gal80ts larvae (24-48 hours),
grown at 17°C, were transferred to 29°C for 2 or 3 days to activate
the Gal4 protein, and then grown at 17°C to suppress Gal4 activity.
Haltere discs were fixed at different times after the temperature change and
GFP and total Ubx protein monitored (hereafter, we refer to this protocol of
temperature change as the standard protocol). We consistently observe three
effects in these discs: first, a progressive decay of GFP and Ubx expression
in the dorsal region, the latter at a faster rate, revealing absence of Gal4
activity after the temperature change (Fig.
3B-D'). Second, the Endo-Ubx expression is not
restored. Even after 6 days at 17°C, when Exo-Ubx is no longer
present, Endo-Ubx transcription has not resumed
(Fig. 3D,D'), indicating
a permanent Ubx repression. In situ hybridization experiments with a
Ubx probe showed that Ubx transcription is similarly
repressed in the dorsal compartment (not shown). Finally, because there is no
Ubx protein in the dorsal compartment of the haltere disc, this is transformed
into the corresponding compartment of the wing disc, and increases its size
significantly (Fig. 3C,D).
These results are not explained by Ubx needing an exceptionally
long time to restore its expression. We used a UAS-dsUbxRNA construct
(Monier et al., 2005) to
prevent Ubx protein synthesis by RNA interference in the ap domain.
If the larvae are kept at 29°C, there is no Ubx protein expression in the
dorsal compartment (Fig. 3E),
but if they undergo our standard protocol, Ubx expression in this
compartment is almost completely restored after 4 days at 17°C
(Fig. 3F). Such recovery is not
observed in ap-Gal4 UAS-GFP/+;
UAS-Ubx/tub-Gal80ts larvae that underwent the
same temperature changes, not even after 6 or 7 days at 17°C
(Fig. 3C-D' and data not
shown). We conclude that a mechanism must prevent the restoration of
Ubx transcription in the latter case.
In accordance with these results, we observe a different phenotype in
halteres of flies expressing Ubx permanently or transitorily. In
ap-Gal4/UAS-Ubx flies, the halteres are reduced
(Fig. 3G), as has been
described previously when Ubx levels are increased
(Smolik-Utlaut, 1990;
Irvine et al., 1993). By
contrast, if this increased expression is transient, a transformation of
haltere into wing ensues (Fig.
3H). Similar phenotypic effects, under similar experimental
regimes, are obtained when we use a scalloped-Gal4 (sd-Gal4)
line, which also drives expression in the haltere disc (not shown). These
experiments explain the contrasting effects obtained with different Gal4 lines
when expressing Ubx. Those that reduce haltere size most probably
maintain a fixed domain of expression throughout development, whereas those
that show transformations of haltere to wing, like dpp-Gal4 or
MS372-Gal4, vary their expression domains with time
(Weigmann and Cohen, 1999)
(data not shown).
The repression of Ubx is maintained cell-autonomously
We wanted to know whether the permanent repression of Ubx by its
own product is a cell autonomous effect. To this aim, we induced clones
expressing transitorily the Ubx product and studied exogenous, endogenous and
total Ubx expression in these clones after several temperature
changes (see Materials and methods). We observe in these clones that
Endo-Ubx expression is continuously repressed in all the cells that
previously expressed Exo-Ubx (even several days after the exogenous
product is no longer present), but not outside it
(Fig. 3I-L). This suggests that
the permanent Ubx repression is maintained cell autonomously.
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We guessed that the different effect of the
Ubx-Gal4SS.2 line may depend on its location within the
Ubx gene. Ubx-Gal4M1 and
Ubx-Gal4M3 have been mapped upstream and close to the
Ubx transcription start site (de
Navas et al., 2006). By contrast, we have located the
Ubx-Gal4SS.2 insertion to position 274.277 (coordinates
according to Martin et al., 1985), very close to the Polycomb
response element (PRE) of the bithorax (bx) region of
Ubx (Orlando et al.,
1998; Ringrose et al.,
2003; Papp and Müller,
2006; Beisel et al.,
2007) (Fig. 4H). We
suspect that the particular position of the Ubx-Gal4SS.2
insertion may account for its different morphological effect when expressing
Ubx.
The permanent repression of Ubx depends on the Pc-group and trx-group genes
The continuous repression of Ubx and the location of the
Ubx-Gal4SS.2 insertion (with its particular properties)
close to the bx PRE suggest that Pc-group genes may be part
of the mechanism used for Ubx permanent repression. To verify this,
we first examined whether the partial transformation of halteres into wings
observed in Ubx-Gal4SS.2 UAS-Ubx flies was
modified in a Polycomb or trithorax heterozygous mutant
background. In Ubx-Gal4SS.2
UAS-Ubx/Pc3 flies there is a significant
reduction in the penetrance (and expressivity) of the haltere to wing
transformation when compared with the controls. An opposite effect is observed
in Ubx-Gal4SS.2 UAS-Ubx/trxE2
flies (Fig. 5A). A second
experiment made use of the UAS-Pcl-RNAi construct, which inactivates
the Polycomblike (Pcl) gene, a member of the Pc
group (Duncan, 1982). We
compared the Ubx expression in third instar haltere discs of
ap-Gal4 UAS-GFP/+; UAS-Ubx
tub-Gal80ts/TM6B and ap-Gal4 UASGFP/+;
UAS-Ubx tub-Gal80ts/UAS-PclRNAi larvae that went
through the standard protocol of temperature changes. The area that lacks Ubx
protein is much larger in the former than in the latter
(Fig. 5B). Finally, we induced
Pc mutant clones in haltere discs where permanent repression of
Ubx had been established (see Materials and methods). In many of
these clones we observed derepression of Ubx, strongly suggesting
that Pc is required to maintain the Ubx silencing induced by
high Ubx levels (Fig.
5C-C'').
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), they
may also cause permanent repression of Ubx in haltere discs. In
MS372-Gal4/UAS-abd-A haltere discs, there are large areas of
Ubx repression that resemble those observed in the same discs of
MS372-Gal4/UAS-Ubx larvae
(Fig. 6A, compare with
Fig. 1F), and
Ubx-Gal4SS.2/UAS-abd-A adults show a strong
transformation of halteres into wings (Fig.
6B). As Abd-A can also make normal halteres
(Casares et al., 1996;
de Navas et al., 2006), these
results strongly suggest that Abd-A can repress Ubx permanently in
haltere discs. A different result is obtained with Abd-B, as in MS372-Gal4/UAS-Abd-B adults there is no or weak transformation of halteres into wings (Fig. 6C). To study in detail whether Abd-B can permanently repress Ubx, we first studied Ubx expression in ap-Gal4 UAS-GFP/UAS-Abd-B(m); tub-Gal80ts/+ haltere discs of larvae grown at 29°C for 3 or more days. In the dorsal compartment of such discs, Ubx signal disappears or is strongly reduced (Fig. 6D-G). By contrast, if larvae of this genotype are grown according to our standard protocol, and the discs fixed 4 days after the last transfer to 17°C, only a small proportion of haltere discs show patches of cells lacking Ubx protein (Fig. 6H). Consistently, only a few flies in this experiment show transformations of halteres into wings. Our conclusion is that Abd-A and Abd-B can repress Ubx transcription in haltere discs, but that only Abd-A can consistently induce permanent Ubx repression.
Ubx and Abd-A proteins share common protein motifs, like the Hexapeptide
(HX) and the UbdA ones, which the Abd-B protein lacks
(Chan and Mann, 1993;
Bürglin, 1994;
Mann and Chan, 1996). To
investigate whether any of these domains accounts for the differences we
detected between Ubx and Abd-B, we expressed Ubx proteins lacking either the
Hexapeptide (UbxHX) or the Ubd-A
(UbxUbdA) domains
(Merabet et al., 2007), and
analyzed whether there is permanent repression of Ubx. Many
Ubx-Gal4SS.2 UAS-UbxHX flies
show transformations of haltere into wing and, occasionally, of metanotum into
mesonotum (Fig. 6I).
Consistently, in ap-Gal4 UAS-GFP/UAS-UbxHX;
tub-Gal80ts/+ larvae that went through our
standard protocol, we see permanent repression of Ubx signal in the dorsal
compartment of the haltere disc (Fig.
6J). By contrast, in
Ubx-Gal4SS.2/UAS-UbxUbdA
flies, the halteres are not transformed into wings
(Fig. 6K) and there is no
permanent repression of Ubx in ap-Gal4
UAS-GFP/UAS-UbxUbdA; tub-Gal80ts/+
haltere discs that underwent the standard treatment
(Fig. 6L). After 5 days at
29°C, we see suppression of the Ubxlac1 reporter in
most cells of the haltere disc (Fig.
6M) and if the larvae are then transferred to 17°C they give
rise to adults with wild-type halteres (not shown). These results suggest that
the UbdA domain may not be essential for Ubx repression, but for
permanent Ubx silencing. However, the UbxUbdA protein
downregulates, but does not suppress, Ubxlac1 expression
after 3 days at 29°C (not shown), indicating that in our standard protocol
the absence of silencing may be due to lack of complete Ubx
repression. We have also observed that expressing the Ubx protein from the
crustacean Artemia franciscana, with a different C-terminal domain
from that of the Drosophila Ubx protein
(Galant et al., 2002;
Ronshaugen et al., 2002), also
leads to permanent repression of Ubx (in
Ubx-Gal4SS.2 UAS-Ubx-Af flies there is
transformation of halteres into wings). Out of the domains tested, the UbdA
may be the only motif in the Ubx protein required for Ubx permanent
repression.
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;
Simmonds et al., 1995;
Tabata et al., 1995).
Increased expression of the en gene driven by different Gal4 lines
causes transformations of posterior-to-anterior compartments in the notum and
the wing resembling those observed in weak en mutants
(Guillén et al., 1995;
Simmonds et al., 1995;
Tabata et al., 1995)
(Fig. 7A-C); wing discs of
these mutant combinations show elimination of the En protein in large areas
(Guillén et al., 1985; Tabata et
al., 1995) (Fig.
7E, compare with the wild type in
Fig. 7D). We have also observed
that in en-Gal4 UAS-GFP/+; UAS-en/+ wing discs, both the En
and the GFP proteins disappear from the same groups of cells of the posterior
compartment (Fig. 7F,G) (see
also Tabata et al., 1985). This suggests that both the en gene and
the en-Gal4 insertion are permanently repressed by the En product.
All these results point to the En protein being able to permanently repress
its own expression. To confirm this inference, we have looked to En and
en-lacZ expression (which reveals en endogenous expression)
in larvae of the ap-Gal4 UAS-GFP/en-lacZ;
tub-Gal80ts/UAS-En genotype that have been kept 5 days at
29°C before taking them back to 17°C. After 3 days at 17°C, we do
not see recovery of either en-lacZ or En protein expression
in the dorsal posterior compartment of the wing disc
(Fig. 7H-K).
Different results are obtained when studying abd-A and
Abd-B. By using P-lacZ lines as reporters of the expression of these
genes in the genital disc (Bender and
Hudson, 2000; Foronda et al.,
2006) (D.F. and E.S.-H., unpublished), we find no repression of
the endogenous gene by Abd-A, and downregulation, but not suppression, of the
Abd-B reporter expression by the Abd-B product (see Fig. S1 in the
supplementary material).
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Guillén et al., 1995;
Simmonds et al., 1995;
Tabata et al., 1995;
Casares et al., 1997). This is
probably a compensation mechanism to cope with protein level fluctuations
during development, which could interfere with the correct expression of
target genes that are highly sensitive to Ubx concentration
(Tour et al., 2005). High
levels of Ubx in the haltere disc, for example, reduce haltere size
(Smolik-Utlaut, 1990). This
negative regulation demands a precise control of protein levels, which is of
high importance to fly development because, as we have seen, if such
fine-tuning does not take place, the two genes are permanently
inactivated.
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; Hogga
and Karch, 2002; Rank et al.,
2002; Schmitt et al.,
2005). We assume that the phenomenon we have observed
(Pc-dependent permanent repression of Ubx) makes use of a
converse mechanism, that is, assembling of Pc complexes at the PRE
following absence of transcription. There is a PRE located in the large
Ubx intron (Chiang et al.,
1995; Orlando et al.,
1998) and it was suggested that Ubx transcription through
this PRE may contribute to inactivation of Pc-complexes
(Papp and Müller, 2006).
Our results support this view. If Ubx transcription is suppressed, as
it is when we force high Ubx levels, Pc-group complexes may
become active at this PRE and repress the Ubx region
(Fig. 8). How Ubx
transcription may inactivate Pc-complexes activity is not clear. It
has been proposed that transcription through a PRE may prevent binding of the
complexes to the DNA (Bender and
Fitzgerald, 2002; Hogga and
Karch. 2002; Rank et al.,
2002; Schmitt et al.,
2005). However, recent experiments have demonstrated that
Pc-group proteins are bound to the bx PRE in haltere discs,
where Ubx is transcribed (Papp
and Müller, 2006). Nevertheless, binding of some of these
proteins is reduced in the haltere disc, when compared with the wing disc,
suggesting that Ubx transcription may reduce this binding
(Papp and Müller, 2006).
Whether affecting Pc-group proteins binding or activation, our
results favor the view that Ubx transcription is a requisite to
prevent Ubx silencing and that absence of transcription leads to
permanent repression.
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),
thus repressing the Ubx-Gal4SS.2 driver
(Fig. 8). This contrasts with
what happens with other Ubx-Gal4 lines, located far from the PRE
(de Navas et al., 2006) and in
which the inactivation of the driver occurs more rarely. Our hypothesis is
that, in the latter, there is repression of the Gal4 line by the exogenous Ubx
product. This repression reduces Ubx protein levels, relieves endogenous
Ubx repression, and the subsequent Ubx transcription through
the PRE prevents Pc-mediated silencing. It is possible that Ubx does
not repress the Ubx-Gal4SS.2 line as efficiently
as the other Ubx-Gal4 lines owing to its position close to the PRE.
Alternatively, the Pc-group complexes may not completely silence
Ubx-Gal4M1 and Ubx-Gal4M3
insertions.
Although repression of transcription is a requisite for establishing
permanent repression in the Ubx gene, it may not be sufficient. We
have shown that abd-A, but not Abd-B, consistently achieve
Ubx silencing in the haltere disc, although both genes repress
Ubx transcription. This different behavior of Ubx/Abd-A and Abd-B
proteins depends neither on the C-terminal region, which contains a conserved
block of glutamines and alanines (Galant
et al., 2002; Ronshaugen et
al., 2002), nor on the HX motif, but may depend on the presence of
the UbdA domain. The UbxUbdA protein can partially transform wings
into halteres (not shown) and downregulates wing disc-specific targets
(Merabet et al., 2007), but is
unable to establish permanent Ubx repression under our standard
protocol conditions. It is possible that the lack of permanent repression we
observe when expressing Abd-B or UbxUbdA may be
due to these proteins allowing very low levels of Ubx transcription,
enough to prevent Pc-mediated permanent repression. In fact, the
UbxUbdA protein needs to be present for a long time (5 days at
29°C in our experiments) to achieve complete repression of the endogenous
Ubx. Alternatively, the UbdA domain may be necessary for the Ubx
protein to collaborate with the establishment of Pc silencing.
If this requirement of the Ubx protein to establish Pc-dependent
Ubx repression is true, it may be needed only in structures where
Ubx is transcriptionally active, such as the haltere disc. Obviously,
in segments anterior to PS5, repression of Ubx by Pc-group
proteins cannot depend on Ubx. In this context, it is relevant to
mention a specific case of Ubx repression: that occurring in the
posterior wing disc. The Ubx promoter is ectopically expressed in the
posterior compartment of the larval wing disc in mutations (like bx
or abx mutations) that eliminate Ubx expression in this
compartment (Irvine et al.,
1993). This suggests there is Ubx early expression in
this domain that is subsequently shut off by the Ubx protein itself
(Irvine et al., 1993) and that
the repression is maintained by Pc-group genes. We have found there
is indeed Ubx expression in the posterior compartment of the
incipient wing disc (in stage 12 embryos) and that this expression disappears
from the wing disc at later embryonic stages (stage 16; see Fig. S2 in the
supplementary material). Therefore, in the mature posterior wing disc there is
a Pc-dependent permanent repression of Ubx that was set
after a transient Ubx protein expression, similar to the mechanism we have
shown. Such early expression may confer specific properties to cells that
initially express Ubx. Thus, in mutations that inactivate partially a
Pc-group gene (in Pc/+ heterozygous, for example),
ectopic Ubx expression is detected in the posterior compartment of
the wing disc much more frequently than in the anterior one (or in other
anterior discs) (e.g. Cabrera et al.,
1985). This suggests that this compartment is somehow `poised' to
activate Ubx when the conditions of repression are reduced. There is
probably a state in the chromatin that favors Ubx derepression in
such a compartment, and this may be due to the early embryonic expression of
Ubx.
The en gene, like Ubx, is negatively autoregulated in
imaginal discs (Guillén et al.,
1995; Simmonds et al.,
1995; Tabata et al.,
1995) and is also regulated by Pc-group proteins
(Busturia and Morata, 1988;
Moazed and O'Farrel, 1992;
McKeon et al., 1994). There is
a PRE in the en gene located upstream its transcription start site
(Kassis et al., 1991;
Kassis, 1994) and
transcription through this PRE has been reported
(Schmitt et al., 2005). We
have shown that high levels of En protein permanently repress en
expression in the wing disc. Analogous to what we have shown with the
Ubx gene, we suppose that En may repress the expression of the
transcript(s) running through the PRE (or PREs) and trigger a permanent
repression through Pc-group complexes binding to, or being active at,
the PRE. Other genes are also negatively regulated by their own products in
development, such as Drosophila Distal-less
(Gorfinkiel et al., 1997),
labial (Chouinard and Kaufman,
1991), Suppressor of hairless
(Barolo et al., 2000),
brinker (Moser and Campbell,
2005) and trithorax-like
(Bernués et al., 2007),
mouse Six3 (Zhu et al.,
2002), and Xenopus goosecoid
(Danilov et al., 1998). These
examples support the idea that many genes need to maintain stable levels of
expression in development, as overriding this control may have deleterious
effects. It would be interesting to know whether the mechanism we have
described take place also in these genes.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/19/3219/DC1
|
ACKNOWLEDGMENTS |
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REFERENCES |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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