|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 4 April 2007
doi: 10.1242/dev.02844
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Centro de Biología Molecular Severo Ochoa, CSIC and UAM, Cantoblanco, 28049 Madrid, Spain.
* Author for correspondence (e-mail: jmodol{at}cbm.uam.es)
Accepted 26 February 2007
 |
SUMMARY |
|---|
 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key words: tailup, islet, Notum development, EGFR, Dpp, Drosophila
|
INTRODUCTION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Mann and Morata, 2000). The
EGFR signalling pathway plays a major role, as its absence prevents formation
of the notum (Simcox et al.,
1996; Wang et al.,
2000; Zecca and Struhl,
2002b). In the notum anlage, EGFR signalling activates the genes
of the iroquois complex (Iro-C), a cluster of three related homeodomain genes,
araucan (ara), caupolican (caup) and
mirror (mirr), that are conserved from worms to vertebrates
(reviewed by Cavodeassi et al.,
2001; Gómez-Skarmeta et
al., 1996; McNeill et al.,
1997). Since the inactivity of Iro-C changes the developmental
fate of cells within the presumptive notum territory towards wing hinge
(Diez del Corral et al.,
1999), the Iro-C genes are considered to have a `pronotum'
function and their domain of expression in the second instar disc defines the
extent of the notum territory. However, the overexpression of Iro-C genes
imposes a notum differentiation fate on wing cells only under a limited set of
conditions (Aldaz et al., 2003;
Wang et al., 2000). This
suggests that genes other than Iro-C help to specify notum identity.
Dpp signalling is also important for notum development. In the second
instar disc, it defines the distal limit of the notum by repressing Iro-C in
the hinge territory (Cavodeassi et al.,
2002). Later, Dpp signalling effects a medial (proximal) versus
lateral subdivision of the notum. This involves activation of the GATA factor
Pannier (Pnr) and the Friend of GATA factor U-shaped (Ush)
(Cubadda et al., 1997;
Ramain et al., 1993) in the
medial notum territory (Sato and Saigo,
2000; Tomoyasu et al.,
2000). Pnr, probably together with Ush
(Haenlin et al., 1997),
represses Iro-C in this region and permits its specification as medial notum
(Calleja et al., 2000). An
anterior/posterior subdivision is carried out by eyegone
(eyg), a Pax-homeobox gene that is activated by Iro-C and Pnr and
whose expression is confined to the anterior notum by the Dpp and Hedgehog
pathways (Aldaz et al., 2003).
In the absence of eyg, this territory does not develop. Forced
coexpression of eyg and ara imposes an anterior notum
developmental fate on posterior or lateral notum cells and even on wing cells
(Aldaz et al., 2003).
tup encodes a LIM-homeodomain transcription factor that is
implicated in axon pathfinding and neurotransmitter identity
(Thor and Thomas, 1997). A
vertebrate homologue of Tup, Isl1, is required for the proper development of
the pancreas and heart, and the specification of several cell types, among
them the pancreas islet cells and some motoneurons and interneurons (reviewed
by Hobert and Westphal, 2000;
Hunter and Rhodes, 2005).
LIM-HD factors are capable of multiple protein-protein interactions (reviewed
by Bach, 2000;
Hobert and Westphal, 2000). In
many contexts, a central co-factor is Chip (also known as NLI and Ldb), which
homodimerises and assembles a 2LIM-HD-2Chip-2Ssdp hexamer
(reviewed by Matthews and Visvader,
2003). The LIMHD factor allows the complex to interact with DNA
through its homeodomain, and transcriptional activation seems to be mediated
by the Ssdp proteins (Nishioka et al.,
2005). The organisation and regulatory properties of this hexamer
have been mostly characterised for the LIM-HD Apterous (Ap) in the
Drosophila wing (Chen et al.,
2002;
Fernández-Fúnez et al.,
1998; Milán and Cohen,
1999; Rincón-Limas et
al., 2000; van Meyel et al.,
2003). In the third instar wing disc, tup is expressed in
a posterior/central region of the notum territory that overlaps with the
dorsocentral (DC) and scutellar proneural clusters of the
achaete-scute genes (Biryukova and
Heitzler, 2005; Cubas et al.,
1991; Skeath and Carroll,
1991). Recent work (Biryukova
and Heitzler, 2005) has shown that loss of function of
tup promotes the formation of extra scutellar and DC macrochaetae,
whereas overexpression of tup suppresses bristle development. Tup can
physically interact with Pnr and with Chip
(Biryukova and Heitzler, 2005;
van Meyel et al., 1999), both
positive regulators of achaete-scute expression in the DC proneural
cluster (García-García et
al., 1999; Ramain et al.,
2000). Accordingly, tup has been considered a member of
the prepattern genes that control achaete-scute expression
(Biryukova and Heitzler, 2005).
Here we show that, similarly to Iro-C, tup has an earlier `pronotum'
function that is essential to commit cells to notum development. For this
function, Tup most likely forms a complex with Chip and Ssdp. tup and
Iro-C, respectively, activated by the Dpp and EGFR signalling pathways,
cooperate in accomplishing this commitment.
|
MATERIALS AND METHODS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
).
We obtained (see Parks et al.,
2004) a deletion (tupex4) between the
FRT-bearing insertions WHf04735 and XPd03613
(Thibault et al., 2004) that
removes the entire ORF of tup (deletion of the interval
18.856.481-18.877.346 of chromosome 2L, version 4.2 of the annotated D.
melanogaster genome). tup-specific RNAi was produced with a
UAS-tupIR transgene constructed
(Nagel et al., 2002) using an
810 nucleotide fragment of tup cDNA AF145674 (interval 96-906).
y, w embryos were transformed
(Rubin and Spradling, 1982)
using pUChs
2-3 as a transposase source.
Mosaic analyses
To generate clones of cells mutant for tup, y, w, hs-FLP1.22; tup,
FRT40A/CyO males were crossed with either y, w, hs-FLP1.22; ubi-GFP,
FRT40A/CyO or y, w, hs-FLP1.22; P{y+}25F, ck13,
FRT40A/CyO or f, hs-FLP1.22; P{f+}30, ck, FRT40A/CyO females.
Homozygous tup clones were induced at different developmental stages
by heat treatment at 37°C for either 30 or 60 minutes or by activating a
UAS-FLP transgene with pnrMD237-Gal4
(Calleja et al., 2000),
MS248-Gal4 (Cavodeassi et al.,
2002; Sánchez et al.,
1997) or Ubx-Gal4LDN
(de Navas et al., 2006).
Clones null for members of the EGFR pathway were prepared by incubating at
37°C for 60 minutes y, hs-FLP9F, f36a; FRT82B, ubi-GFP,
P{f+}87D, M(3)95A/FRT82B,
Ras85DC40b or y, hs-FLP9F,
f36a; FRT82B, ubi-GFP, P{f+}87D, M(3)95A/FRT82B,
pnt88 or y, w, hs-FLP1.22;
FRT42D, arm-lacZ, M(2)l2/FRT42D, Egfr1K35 (Egfrf2)
larvae. The M+ genotype
(Morata and Ripoll, 1975) of
the clones was a requisite for their substantial growth. Clones mutant for
Chip or Ssdp were obtained from y, w, hs-FLP1.22;
FRT42D, ubi-GFP/FRT42D, Chie5.5 or y, hs-FLP9F,
f36a; FRT82B, ubi-GFP, P{f+}87D, M(3)95A/FRT82B,
Ssdpneo48, e larvae which were treated at 37°C for 75
minutes.
Overexpression analyses
DC-lacZ/CyO; C765-Gal4 or
dppblk-Gal4/SM6a-TM6b/DC-lacZ females
(García-García et al.,
1999; Gómez-Skarmeta et
al., 1996; Staehling-Hampton
et al., 1994) were crossed to either UAS-ara
(Gómez-Skarmeta et al.,
1996), UAS-tup (Thor
and Thomas, 1997),
UAS-tupHD
(O'Keefe et al., 1998),
UAS-ara; UAS-tup or UAS-ara;
UAS-tupHD males, and the progeny
raised at 25°C. One or two copies of UAS-tupIR were
overexpressed with the MS248-Gal4 driver at 29°C. To overexpress
Mkp3 or Dad during notum specification, males homozygous for
either the UAS-bearing P-GS insertion Mkp3M76
(Ruiz-Gómez et al.,
2005) or the UAS-Dad transgene
(Tsuneizumi et al., 1997) were
crossed with ptc559.1-Gal4,
UAS-GFP/SM6a-TM6b/tub-Gal80ts females
(McGuire et al., 2003;
Speicher et al., 1994).
Progeny were raised at 17°C until mid- or late-second instar, then
switched to 29°C for at least 24 hours and dissected. Clones of cells
overexpressing diverse UAS-X transgenes were generated by incubating
at 34°C for 15 minutes y, w, hs-FLP1.22; Act>y+>Gal4, UAS-GFP/+
UAS-X/+ larvae. Other UASactivated transgenes were: UAS-Chip
(Milán and Cohen,
1999), UASChipDD
(van Meyel et al., 1999),
UAS-tkvQD (Das et al.,
1998), UASRas1V12
(Karim and Rubin, 1998),
UAS-RafDN (Baek et al.,
1996) and UASargos
(Howes et al., 1998).
Antibody staining
Imaginal discs were fixed and stained as described previously
(Cubas et al., 1991).
Antibodies were: mouse anti-Tup (mAb 40.3A4, DSHB), rabbit
anti-ß-galactosidase (Cappel), rat anti-Ara/Caup
(Diez del Corral et al.,
1999), rabbit anti-Msh
(McDonald et al., 1998)
(provided by C. Doe), rabbit anti-Tsh (Ng
et al., 1996), rat anti-Zfh2
(Whitworth and Russell, 2003),
rabbit anti-Ush (Fossett et al.,
2001), guinea pig anti-Eyg
(Aldaz et al., 2003), mouse
anti-Nub (Averof and Cohen,
1997), rabbit anti-Sal (de
Celis et al., 1999). Secondary antibodies and rhodamine phalloidin
were obtained from Molecular Probes or Jackson ImmunoResearch.
Image acquisition
Adult unmounted flies were photographed with a Zeiss Axiophot microscope.
Images of different focal planes were combined using Photoshop (Adobe).
Fluorescence images were captured using a confocal system.
|
RESULTS |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Butler et al., 2003). A
quantitative summary of this phenotypic analysis, comprising over 1600
homozygous tupex4 clones, is presented in Table S1 (see
Table S1 in the supplementary material). Similar phenotypes were observed with
the other tup alleles.
Clones were associated with a variety of phenotypes whose nature and
frequency depended on the position of the clone (see Fig. S1C in the
supplementary material) and on the developmental time of its induction (see
Table S1 in the supplementary material). They ranged from partial or complete
loss of a heminotum (see Fig. S1A in the supplementary material), to formation
on the notum of ectopic wing-hinge structures, malformations of the notum
cuticle (Fig. 1) and
modifications to the bristle pattern. This latter phenotype will not be
described, as effects of tup mutations on this pattern have already
been reported (Biryukova and Heitzler,
2005). The ectopic hinge structures were tegulae
(Fig. 1C) or tegula-like
structures (Fig. 1A,B),
recognisable sclerites (Fig.
1B) and hinge-like sensilla campaniformia
(Fig. 1G,L) or trichoidea (see
Fig. S1B in the supplementary material). Seemingly parallel transformations
occurred on the metathorax, a derivative of the haltere disc, in which
tup is also expressed during larval development (data not shown).
Sensilla campaniformia similar to those found in the basal part of the haltere
were present in the metanotum (Fig.
1D), a region that does not harbour sensilla in the wild type.
Other malformations of the notum cuticle consisted of invaginations (Fig. 1F-I) or protrusions (Fig. 1E). Some invaginations gave rise to vesicles that displayed trichomes and hinge-like sensilla campaniformia (Fig. 1G). At late clone-induction times, a proportion of the vesicles were separated from the notum cuticle, lacked any kind of sensillum, but conserved trichomes (data not shown). Additional morphologically distinct malformations consisted of small, tubercle-like disruptions of the cuticle, with a corrugated appearance and roundish contour (Fig. 1J-L). At their centre, they could have shallow depressions (Fig. 1L) or deep and narrow invaginations (Fig. 1K). The presence of macro- and/or microchaetae indicated that the malformations still developed a notum-like cuticle (Fig. 1E,H,I,K), although occasionally we observed sensilla campaniformia (Fig. 1L) or trichoidea (see Fig. S1 in the supplementary material). The invaginations, projections, tubercles, and attached and detached vesicles probably form a related group of lesions caused by a tendency of tup clones to detach from the notum epidermis, an indication of differential cellular adhesive properties. In summary, a proportion of tup clones give rise to structures indicative of notum-to-hinge transformations, whereas other clones induce malformations suggestive of modified cell-cell adhesion properties, but maintaining a notum-like identity.
|
) and was
seen to extend into the lateral notum (Fig.
2C). In the mid-late third instar, strong expression was
maintained in the posterior medial (arrow) and part of the lateral (arrowhead)
notum (Fig. 2D). Weak residual
activity might be present in the anterior notum
(Fig. 2D, asterisk). Comparison
with ara/caup, which at these stages are expressed in the lateral
notum, indicates that the most lateral region of the posterior notum is
essentially free of Tup (Fig.
2D) (see also Biryukova and
Heitzler, 2005; Butler et al.,
2003).
tup clones show differential affinity in wing discs
We examined the morphology of tup clones in the notum region of
third instar wing discs. Clones induced at the first instar were generally
large and with a smooth border, which at times was associated with an ectopic
fold of the notum epithelium (Fig.
3A). Smaller, later-induced clones, could have either smooth and
roundish, or wiggly borders (Fig.
3B). The smooth clones were more prevalent in the posterior notum,
which is the region of strong tup expression
(Fig. 2D). Smooth contours
suggest a differential affinity between two cell populations, as these tend to
minimise contacts. In addition, many roundish tup clones partially
extruded themselves towards the subjacent adepithelial cells
(Fig. 3C,D). This behaviour
might correlate with the invaginations associated with the adult tup
mutant epidermis. Still, at these stages, clone cells did not lose their
apical connections with the neighbouring wild-type cells, as revealed by the
continuous band of apical actin accumulation
(Fig. 3D).
Notum tup clones express hinge markers
Next, we analysed the expression of hinge markers in discs harbouring
tup clones. msh (also known as Drop - Flybase),
which is expressed strongly at the dorsal hinge and weakly in part of the
posterior notum (D'Alessio and Frasch,
1996; Villa-Cuesta and
Modolell, 2005) (Fig.
3B'', asterisk; Fig.
3G), was always upregulated in first instar-induced clones located
at the medial and central notum (Fig.
3F), in some cases even in the neighbouring wild-type tissue
(Fig. 3E). However, many clones
located at the lateral-most notum failed to upregulate msh. In
later-induced clones, derepression was generally limited to clones at or near
the expression domain of tup. Moreover, the levels of expression were
different from clone to clone (Fig.
3B,B'') and at times even among cells of the same clone
(Fig. 3B''). Qualitatively
similar observations were made with zfh2, which is expressed almost
exclusively in the distal hinge (Whitworth
and Russell, 2003) (Fig.
6L), spalt (sal; also known as salm -
Flybase), which is expressed at high levels in the hinge and lateral notum
territories and at a lower level in the posterior notum
(de Celis et al., 1999)
(Fig. 3I), and the
lacZ insertion line l(2)09261, which is expressed in the
hinge and wing pouch territories (Diez del
Corral et al., 1999). As examples, we show early-induced clones in
which l(2)09261 and sal were respectively upregulated
(Fig. 3A,H), and one clone out
of several expressing msh that also expressed zfh2
(Fig. 3B,B').
|
|
tup clones lose notum markers
Next, we examined the effect of tup clones on genes important for
notum development. pnr expression was removed in all first
instarinduced clones (Fig. 4B),
and also in most later-induced clones (
85%;
Fig. 4E shows exceptions),
especially in those located at the more distal part of the pnr domain
(Fig. 4D). Ush, which
accumulates in a region nested within the pnr domain
(Fig. 4A), was removed in first
and second instar-induced tup clones
(Fig. 4C and not data shown),
and was partially lost in third instar-induced clones. However, in large first
instar-induced clones, ush was often expressed in a subregion of the
clone. This subregion coexpressed msh (data not shown) and usually
displayed a fold of the epithelium (Fig.
4B; see also 4I). These characteristics indicate a transformation
towards hinge, as ush is normally expressed in the hinge region of
the disc that is transversed by several folds
(Fig. 4A). eyg
expression (Fig. 4I, inset) was
lost from first instar-induced tup clones
(Fig. 4I), but not from
later-induced clones.
|
). We examined whether Iro-C products were lost in
tup clones. Loss of Ara/Caup occurred only in a small area of the
central notum (Fig. 4E-G), a
region different from that where hinge structures most often arise (the
lateral notum, Fig. 1A-C and
see Fig. S1C in the supplementary material). Moreover, in the medial notum,
tup clones frequently activated ara/caup
(Fig. 4D,E), an effect probably
resulting from the loss of Pnr (Calleja et
al., 2000) and/or Ush, as the heterodimer Pnr-Ush
(Haenlin et al., 1997) appears
to be a repressor of Iro-C (Letizia et
al., 2007).
Iro-C downregulates msh in the notum territory
(Villa-Cuesta and Modolell,
2005), so the stimulation of msh in tup cells
that did not express Iro-C (Fig.
4G) was expected. However, msh could also be upregulated
in clones in which ara/caup were expressed
(Fig. 4H). Thus, in some
instances, tup cells simultaneously expressed hinge and notum
genes.
Chip and Ssdp are co-factors of Tup for notum specification
Since Tup can physically interact with Chip
(Biryukova and Heitzler, 2005;
van Meyel et al., 1999), we
examined whether this co-factor was involved in the `pronotum' function of
Tup. This seemed to be the case. First instar-induced
Chipe5.5 clones located in the presumptive notum showed
derepression of zfh2 and downregulation of eyg
(Fig. 5A), which indicated a
notum-to-hinge transformation. Moreover, msh was also derepressed in
part of the clones, but only in a non-autonomous manner
(Fig. 5B). [Chip is required
for msh expression in the hinge
(Villa-Cuesta and Modolell,
2005), so the absence of msh activation within the clones
was expected.] Some of the flies bearing Chip clones survived to
adulthood and showed cuticular defects similar to those associated with
early-induced tup clones, including ectopic tegulae and sensilla
trichoidea (see Fig. S2B in the supplementary material).
As the above results indicate that Tup and Chip are both positive effectors
of notum specification, and given that they can physically interact
(Biryukova and Heitzler, 2005;
van Meyel et al., 1999), we
asked whether they might function as an hexameric complex with Ssdp, similar
to the 2Ap-2Chip-2Ssdp complex (reviewed by
Matthews and Visvader, 2003).
We tested whether Ssdp affected notum specification. We used the
hypomorphic Ssdpneo48 allele, as clones null for
Ssdp are not recovered in adults
(van Meyel et al., 2003) and
hardly grow in imaginal discs even in a Minute heterozygous
background (data not shown). Forty per cent of Ssdpneo48
clones lost eyg expression and gained zfh2 expression
(Fig. 5C), and adult flies
bearing these clones showed cuticular defects similar to those harbouring
tup or Chip clones (see Fig. S2C in the supplementary
material) and, in one example, showed an outgrowth composed of proximal costa
tissue (see Fig. S2D,E in the supplementary material).
In the wing, an experimental excess of Chip titrates Ap and Ssdp, prevents
formation of the hexameric complex, and phenotypically mimics the
loss-of-function of Chip
(Fernández-Fúnez et al.,
1998; Milán and Cohen,
1999; Rincón-Limas et
al., 2000). Accordingly, we checked whether an excess of Chip also
interfered with notum specification. First instar-induced clones
overexpressing either UASChip or
UAS-ChipDD (which lacks the
dimerisation domain) in the posterior and proximal notum showed loss of
eyg expression and acquired expression of zfh2
(Fig. 5D and data not
shown).
Overexpression of tup and ara synergistically promote notum development
We compared the ability of tup and the Iro-C gene ara,
overexpressed either singly or together, to impose notum development on cells
normally fated to differentiate into other structures. Ubiquitous, relatively
late overexpression of UAS-tup (C765-Gal4 driver)
(Gómez-Skarmeta et al.,
1996) induced formation of notum-like tissue in the mesopleura
(Fig. 6A,C) and extra
notum-like bristles on the tegula (Fig.
6C). By contrast, overexpression of UAS-ara under the
same conditions did not induce notum-like structures
(Fig. 6B), although it reduced
the size of the wing (see
Gómez-Skarmeta et al.,
1996). Overexpression of both UAS-ara and
UAS-tup had a more drastic effect: the wing and wing hinge were
replaced by a large structure of notum-like tissue
(Fig. 6D). The notum-like
structure was also present on the mesopleura, a territory where Iro-C is
expressed in the wild type
(Gómez-Skarmeta et al.,
1996). None of these effects were observed (data not shown) upon
overexpression of a truncated Tup protein lacking the homeodomain
(UAS-tupHD).
|
). Coexpression of UAS-tup and UAS-ara
greatly expanded the area of expression of eyg to parts of the dorsal
hinge, the ventral hinge, pleura and wing pouch.
(Fig. 6H), consistent with the
formation of large, notum-like structures.
Overexpression of both UAS-ara and UAS-tup with
dpp-Gal4, which drives expression in a central stripe of the wing
pouch (Staehling-Hampton et al.,
1994), transformed the central part of the wing to notum-like
tissue (Fig. 6I), whereas the
anterior and posterior parts developed as wing tissue. Consistently in this
phenotype, eyg was upregulated in the overexpression territory
(Fig. 6K), whereas Zfh2 and the
wing pouch marker Nub (Ng et al.,
1995) were lost (Fig.
6L,M, arrowheads). Moreover, this driver also directs expression
in leg discs, and eyg was derepressed in the sternopleural region
(Fig. 6J). The adults displayed
notum-like structures near the coxa (Fig.
6I), which indicated a transformation of this ventral region of
the body wall towards notum. This transformation was not observed when
UAS-ara or UAS-tup were overexpressed singly. Taken
together, these results suggest a synergism of Iro-C and tup in
promoting notum development.
tup and Iro-C are differently regulated
In the notum territory, Iro-C is activated by the EGFR signalling pathway
(Wang et al., 2000;
Zecca and Struhl, 2002a). This
led us to examine whether tup was also controlled by EGFR. Clones
homozygous for the null Egfr1K35 allele suppressed
expression of ara/caup as expected, but not that of tup
(Fig. 7A). Similar results were
obtained with
Ras85DC40b
(Fig. 7B) or
pnt88 clones, or by
overexpressing UAS-argos or UAS-RafDN
(Raf is also known as phl - Flybase) (data not shown), all
of which constitute milder conditions for inhibiting the EGFR pathway.
Moreover, constitutive activation of the EGFR pathway by overexpressing
UAS-Ras1V12, clearly activated ara/caup in the
hinge territory, but not so tup
(Fig. 7E). Similar clones in
the notum did not modify tup expression. The independence of
tup from the EGFR pathway was also verified at developmental times
close to those of notum specification
(Wang et al., 2000). In second
and early third instar wing discs, overexpression of Mkp3, a strong
inhibitor of the pathway
(Ruiz-Gómez et al.,
2005), reduced notum growth and clearly inhibited
ara/caup, whereas tup remained almost unaffected
(Fig. 7C,D). Together, these
data strongly argue against any control of tup by EGFR.
Dpp signalling negatively regulates Iro-C and restricts its expression to
the lateral notum (Cavodeassi et al.,
2002). By contrast, removal of Dpp signalling in
tkva12 clones suppressed tup expression
(Fig. 7F), except in some of
the clones located in the lateral-most region. Moreover, overexpression of
Dad, a strong inhibitor of the Dpp pathway
(Tsuneizumi et al., 1997),
turned off tup in second and early third instar discs
(Fig. 7G). Conversely,
activation of the Dpp pathway by the overexpression of
UAS-tkvQD, upregulated tup in the medial notum,
although not so in the lateral notum (Fig.
7H). We conclude that Dpp signalling is a principal positive
regulator of tup, although additional regulators probably exist and
should account for the expression of tup in the Dppinsensitive
regions. Hence, Iro-C and tup appear to be differently regulated in
this disc.
|
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). Here we show that tup functions earlier in the
development of the dorsal mesothorax. Loss of tup causes a range of
phenotypes, which taken together indicate interference with the assignment of
cells to form notum. Thus, depending on the time of induction of the clones
and their location, we observe the formation of notum-like cuticle with
altered cell-cell adhesion properties, the generation of ectopic wing-hinge
structures including tegulae, sclerites or sensilla typical of the proximal
wing, or even the loss of the entire heminotum. Consistent with these adult
phenotypes, in third instar wing discs tup mutant cells can
upregulate genes typically expressed at high levels in the wing-hinge
territory of the disc, such as zfh2, msh, sal and the lacZ
insertion line l(2)09261. Concomitantly, notum-expressed genes such
as eyg, ush and pnr are generally repressed, although in
some cases tup cells may abnormally express notum and hinge genes
together. These data indicate that notum tup cells undergo
transformation towards either an altered notum fate or a hinge fate. Moreover,
the activation of hinge markers in wild-type cells surrounding some
tup clones might reflect the presence of ectopic notum/hinge borders,
which are known to promote non-autonomous effects
(Diez del Corral et al., 1999;
Villa-Cuesta and Modolell,
2005).
|
;
Hunter and Rhodes, 2005), Tup
is required for the proper specification of not only cell types
(Biryukova and Heitzler, 2005;
Thor and Thomas, 1997), but
also developing territories.
Tup associates with Chip and Ssdp for notum specification
Tup is known to bind the co-factor Chip
(Biryukova and Heitzler, 2005;
van Meyel et al., 1999).
Since, in dorsal compartment specification, Chip functions in a
2Ap-2Chip-2Sspd hexamer, we asked whether a similar 2Tup-2Chip-2Sspd complex
might mediate Tup function in notum specification. Our results support this
interpretation. The loss of either Chip or Ssdp upregulated hinge genes
(zfh2, msh), repressed a notum marker (eyg), and induced
cuticular defects similar to those associated with tup clones.
Moreover, an excess of Chip would be expected to titrate Tup and/or Ssdp in
incomplete complexes and mimic the loss-of-function phenotype of
notum-to-hinge transformation, as was experimentally observed.
By contrast, during the later process of sensory organ formation, Tup
appears to act by sequestering both Chip and Pnr, thus preventing activation
of the proneural genes achaete-scute
(Biryukova and Heitzler, 2005).
This negative function of Tup does not seem relevant for notum specification,
where both Tup and Chip work as positive effectors. Moreover, the Tup
homeodomain is dispensable for titrating Chip and Pnr
(Biryukova and Heitzler, 2005),
but this is not the case for its `pronotum' function (J.deN., unpublished).
Interestingly, a missense mutation within the LIM-interacting interacting
domain of Chip (ChipE) severely reduces its ability to
interact with Tup and suppresses the negative regulation by Tup of bristle
formation (Biryukova and Heitzler,
2005). However, homozygous ChipE flies have no
defects in notum specification (Ramain et
al., 2000). This suggests that a residual interaction between
ChipE and Tup might persist, as additionally suggested by the
suppression of the extra bristles present in ChipE
individuals by UAS-tup overexpression
(Biryukova and Heitzler, 2005).
A weak interaction between Tup and Chip, which might only permit the formation
of low levels of hexameric complex, might still allow proper notum
specification. This suggestion agrees with the fact that
tupd03613, a strong hypomorphic allele (as substantiated
by its embryonic lethality over the null tupex4; J.deN.,
unpublished), allows proper notum formation in homozygosis
(Biryukova and Heitzler,
2005).
|
). By contrast, loss of tup causes an assortment of
different combinations of derepressed hinge genes and repressed notum genes.
Moreover, many tup clones induced during the second larval instar,
and even some induced in the first, can develop recognisable notum cuticle.
Thus, we propose that tup reinforces/stabilises the commitment of
cells to develop as notum, a commitment imposed mainly by Iro-C. This
reinforcement or stabilisation might be most necessary in the proximal part of
the disc, where expression of ara/caup ceases after the second
instar, but that of tup persists. This might account for the
derepression of hinge genes being most manifest in this region. Depending on
the location and time of Tup deprival, its loss may be inconsequential or lead
to a partial or even a complete loss of notum commitment. Such diversity of
consequences led us to explore whether tup might act on target genes
by affecting chromatin remodelling. However, no genetic interactions have been
found with Polycomb (Pc, Scr+Pcl+esc) or trithorax
(trx, osa, brm, Trl, lawc) group genes (J.deN., unpublished).
In contrast to the absolute requirement for Iro-C for notum specification,
overexpression of UAS-ara can impose a notum fate only on the wing
anlage, and only when provided early in the development of the disc
(Aldaz et al., 2003;
Wang et al., 2000) (R. Diez
del Corral, PhD thesis, Universidad Autónoma de Madrid, 1998). An extra
notum with mirror-image disposition versus the extant notum is generated at
the expense of the wing, a phenotype identical to that resulting from early
deprivation of Wg function (Couso et al.,
1993; Morata and Lawrence,
1977; Ng et al.,
1996; Sharma and Chopra,
1976). As UAS-ara overexpression can interfere with
wg expression (R. Diez del Corral, PhD thesis, Universidad
Autónoma de Madrid, 1998), Wg deprival probably explains the formation
of the extra notum. Thus, by itself, overexpression of UASara
probably lacks a genuine potential for imposing the notum fate. Similar notum
duplications arise upon early and strong overexpression of UAS-tup
(MD638, dpp-Gal4 and ptc-Gal4 drivers) and, again, they
probably result from inhibition of Wg activity (J.deN., unpublished).
Consistent with this interpretation, weaker and later expression of either
UAS-tup or UAS-ara (C765 driver)
(Gómez-Skarmeta et al.,
1996) has little or no capacity to promote notum fate. However,
when coexpressed, these transgenes are effective in imposing the notum fate
and this should not be attributed to Wg depletion. Indeed, the transformation
consists of an expansion of the notum tissue
(Fig. 6D), rather than a notum
duplication (Morata and Lawrence,
1977). Moreover, as detected by the onset of the ectopic
expression of notum markers (eyg, DC-lacZ), the transformation occurs
in late third instar discs (J.deN., unpublished) that have a nearly wild-type
morphology and a distinguishable wing pouch
(Fig. 6H). This indicates that
these markers are activated in territories previously specified as wing, hinge
or pleura, and subsequently forced to acquire notum identity. Moreover,
overexpression of the Wg pathway antagonists UAS-Axin or
UAS-dTCFDN (dTCF is also known as pan -
Flybase) with the same driver failed to transform wing towards notum (J.deN.,
unpublished). Finally, the activation of eyg and the formation of
notum tissue in the sternopleurite, a derivative of the leg disc, also attest
to the capacity of tup plus ara to commit cells to develop
as notum.
EGFR and Dpp signalling pathways collaborate in notum specification
It is well established that signalling by the EGFR pathway is essential for
notum development. Its inhibition prevents activation of Iro-C and the growth
of the notum territory (Simcox et al.,
1996; Wang et al.,
2000; Zecca and Struhl,
2002b). By contrast, Dpp negatively regulates Iro-C and restricts
its domain of expression at both its distal and proximal borders
(Cavodeassi et al., 2002). Our
data indicate a novel function of Dpp in notum development; namely, the
activation or maintenance of tup expression in second and third
instar discs. In the notum region of the early disc, Dpp signalling occurs at
low levels (Cavodeassi et al.,
2002), but our results suggest that these are sufficient for
activating tup. Expression of tup is largely independent on
EGFR signalling. Thus, EGFR and Dpp signalling seem to cooperate in specifying
notum identity to the cells of the proximal part of the disc by activating
their respective `pronotum' downstream genes, Iro-C and tup.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/9/1779/DC1
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Aldaz, S., Morata, G. and Azpiazu, N. (2003).
The Pax-homeobox gene eyegone is involved in the subdivision of the
thorax of Drosophila. Development
130,4473
-4482.
Averof, M. and Cohen, S. M. (1997). Evolutionary origin of insect wings from ancestral gills. Nature 385,627 -630.[CrossRef][Medline]
Bach, I. (2000). The LIM domain: regulation by association. Mech. Dev. 91, 5-17.[CrossRef][Medline]
Baek, K. H., Fabian, J. R., Sprenger, F., Morrison, D. K. and Ambrosio, L. (1996). The activity of D-raf in torso signal transduction is altered by serine substitution, Nterminal deletion, and membrane targeting. Dev. Biol. 175,191 -204.[CrossRef][Medline]
Biryukova, I. and Heitzler, P. (2005). The Drosophila LIM-homeodomain protein Islet antagonizes pro-neural cell specification in the peripheral nervous system. Dev. Biol. 288,559 -570.[CrossRef][Medline]
Butler, M. J., Jacobsen, T. L., Cain, D. M., Jarman, M. G.,
Hubank, M., Whittle, J. R., Phillips, R. and Simcox, A.
(2003). Discovery of genes with highly restricted expression
patterns in the Drosophila wing disc using DNA oligonucleotide
microarrays. Development
130,659
-670.
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.[Abstract]
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]
Cavodeassi, F., Modolell, J. and Gómez-Skarmeta, J.
L. (2001). The Iroquois family of genes: from body building
to neural patterning. Development
128,2847
-2855.
Cavodeassi, F., Rodríguez, I. and Modolell, J. (2002). Dpp signalling is a key effector of the wing-body wall subdivision of the Drosophila mesothorax. Development 129,3815 -3823.[Medline]
Chen, L., Segal, D., Hukriede, N. A., Podtelejnikov, A. V.,
Bayarsaihan, D., Kennison, J. A., Ogryzko, V. V., Dawid, I. B. and Westphal,
H. (2002). Ssdp proteins interact with the LIM-domain-binding
protein Ldb1 to regulate development. Proc. Natl. Acad. Sci.
USA 99,14320
-14325.
Couso, J. P., Bate, M. and Martínez-Arias, A.
(1993). A wingless-dependent polar coordinate system in
Drosophila imaginal discs. Science
259,484
-489.
Cubadda, Y., Heitzler, P., Ray, R. P., Bourouis, M., Ramain, P.,
Gelbart, W., Simpson, P. and Haenlin, M. (1997).
u-shaped encodes a zinc finger protein that regulates the proneural
genes achaete and scute during formation of bristles in
Drosophila. Genes Dev.
11,3083
-3095.
Cubas, P., de Celis, J. F., Campuzano, S. and Modolell, J.
(1991). Proneural clusters of achaete-scute expression
and the generation of sensory organs in the Drosophila imaginal wing
disc. Genes Dev. 5,996
-1008.
D'Alessio, M. and Frasch, M. (1996). msh may play a conserved role in dorsoventral patterning of the neuroectoderm and mesoderm. Mech. Dev. 58,217 -231.[CrossRef][Medline]
Das, P., Maduzia, L. L., Wang, H., Finelli, A. L., Cho, S. H., Smith, M. M. and Padget, R. W. (1998). The Drosophila gene Medea demonstrates the requirement for different classes of Smads in dpp signaling. Development 125,1519 -1528.[Abstract]
de Celis, J. F., Barrio, R. and Kafatos, F. (1999). Regulation of the spalt/spaltrelated gene complex and its function during sensory organ development in the Drosophila thorax. Development 126,2653 -2662.[Abstract]
de Navas, L., Foronda, D., Suzanne, M. and Sánchez-Herrero, E. (2006). A simple and efficient method to identify replacements of P-lacZ by P-Gal4 lines allows obtaining Gal4 insertions in the bithorax complex of Drosophila.Mech. Dev. 123,860 -867.[CrossRef][Medline]
Diez del Corral, R., Aroca, P., Gómez-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.
Fernández-Fúnez, P., Lu, C. H., Rincón-Limas, D. E., García-Bellido, A. and Botas, J. (1998). The relative expression amounts of apterous and its co-factor dLdb/Chip are critical for dorso-ventral compartmentalization in the Drosophila wing. EMBO J. 17,6846 -6853.[CrossRef][Medline]
Fossett, N., Tevosian, S. G., Gajewski, K., Zhang, Q., Orkin, S.
H. and Schulz, R. A. (2001). The Friend of GATA proteins
U-shaped, FOG-1, and FOG-2 function as negative regulators of blood, heart,
and eye development in Drosophila. Proc. Natl. Acad. Sci.
USA 98,7342
-7347.
García-García, M. J., Ramain, P., Simpson, P. and Modolell, J. (1999). Different contributions of pannier and wingless to the patterning of the dorsal mesothorax of Drosophila. Development 126,3523 -3532.[Abstract]
Gómez-Skarmeta, J. L., Diez del Corral, R., de la Calle-Mustienes, E., Ferrés-Marcó, 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.[CrossRef][Medline]
Haenlin, M., Cubadda, Y., Blondeau, F., Heitzler, P., Lutz, Y.,
Simpson, P. and Ramain, P. (1997). Transcriptional activity
of Pannier is regulated negatively by heterodimerization of the GATA
DNA-binding domain with a cofactor encoded by the u-shaped gene of
Drosophila. Genes Dev.
11,3096
-3108.
Hobert, O. and Westphal, H. (2000). Functions of LIM-homeobox genes. Trends Genet. 16, 75-83.[CrossRef][Medline]
Howes, R., Wasserman, J. D. and Freeman, M.
(1998). In vivo analysis of Argos structure-function.
Sequence requirements for inhibition of the Drosophila epidermal
growth factor receptor. J. Biol. Chem.
273,4275
-4281.
Hunter, C. S. and Rhodes, S. J. (2005). LIM-homeodomain genes in mammalian development and human disease. Mol. Biol. Rep. 32,67 -77.[CrossRef][Medline]
Karim, F. D. and Rubin, G. M. (1998). Ectopic expression of activated Ras 1 induces hyperplastic growth and increased cell death in Drosophila imaginal tissues. Development 125,1 -9.[Abstract]
Letizia, A., Barrio, R. and Campuzano, S.
(2007). Antagonistic and cooperative actions of the EGFR and Dpp
pathways on the iroquois genes regulate Drosophila
mesothorax specification and patterning. Development
134,1337
-1346.
Mann, R. S. and Morata, G. (2000). The developmental and molecular biology of genes that subdivide the body of Drosophila. Annu. Rev. Cell Dev. Biol. 16,243 -271.[CrossRef][Medline]
Matthews, J. M. and Visvader, J. E. (2003). LIM-domain-binding protein 1, a multifunctional cofactor that interacts with diverse proteins. EMBO Rep. 4,1132 -1137.[CrossRef][Medline]
McDonald, J. A., Holbrook, S., Isshiki, T., Weiss, J. B., Doe,
C. Q. and Mellerick, D. M. (1998). Dorsoventral patterning in
the Drosophila central nervous system: the vnd homeobox gene
specifies ventral column identity. Genes Dev.
12,3603
-3612.
McGuire, S. E., Le, P. T., Osborn, A. J., Matsumoto, K. and
Davis, R. L. (2003). Spatiotemporal rescue of memory
dysfunction in Drosophila. Science
302,1765
-1768.
McNeill, H., Yang, C. H., Brodsky, M., Ungos, J. and Simon, M.
A. (1997). mirror encodes a novel PBX-class
homeoprotein that functions in the definition of the dorso-ventral border of
the Drosophila eye. Genes Dev.
11,1073
-1082.
Milán, M. and Cohen, S. M. (1999). Regulation of LIM homeodomain activity in vivo: a tetramer of dLDB and Apterous confers activity and capacity for regulation by dLMO. Mol. Cell 4,267 -273.[CrossRef][Medline]
Morata, G. and Ripoll, P. (1975). Minutes: mutants of Drosophila autonomously affecting cell division rate. Dev. Biol. 42,211 -221.[CrossRef][Medline]
Morata, G. and Lawrence, P. A. (1977). The development of wingless, a homeotic mutation of Drosophila.Dev. Biol. 56,227 -240.[CrossRef][Medline]
Nagel, A. C., Maier, D. and Preiss, A. (2002). Green fluorescent protein as a convenient and versatile marker for studies on functional genomics in Drosophila. Dev. Genes Evol. 212, 93-98.[CrossRef][Medline]
Ng, M., Díaz-Benjumea, F. J. and Cohen, S. M. (1995). nubbin encodes a POUdomain protein required for proximal-distal patterning in the Drosophila wing. Development 121,589 -599.[Abstract]
Ng, M., Díaz-Benjumea, F. J., Vincent, J. P., Wu, J. and Cohen, S. M. (1996). Specification of the wing by localized expression of the wingless protein. Nature 381,316 -318.[CrossRef][Medline]
Nishioka, N., Nagano, S., Nakayama, R., Kiyonari, H., Ijiri, T.,
Taniguchi, K., Shawlot, W., Hayashizaki, Y., Westphal, H., Behringer, R. R. et
al. (2005). Ssdp1 regulates head morphogenesis of mouse
embryos by activating the Lim1-Ldb1 complex.
Development 132,2535
-2546.
O'Keefe, D. D., Thor, S. and Thomas, J. B. (1998). Function and specificity of LIM domains in Drosophila nervous system and wing development. Developmen
