Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Cantoblanco, 28049 Madrid, Spain
E-mail: irodriguez{at}cbm.uam.es
Accepted 19 March 2004
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SUMMARY |
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Key words: Drosophila melanogaster, dachsous, Wg signaling, Pattern formation, Imaginal disc, PD axis
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Introduction |
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The Wnt-ligand controlling PCP still remains to be characterized.
Nevertheless, it is known that the asymmetrical localization of Fz at the
plasma membrane is the response to a polarity signal that is distributed as a
gradient within the epithelium. Members of the cadherin family have been
implicated at different steps in PCP signaling
(Adler, 2002). Cadherins form a
family of well-conserved transmembrane molecules with common structural
features, such as an extracellular domain containing several copies of a
cadherin motif, a transmembrane region, and a cytoplasmatic tail that, in most
cases, binds other proteins, such as
- and ß-catenin
(Vleminckx and Kemler, 1999
).
They are located at adherens junctions and mediate cell-cell adhesion through
homophilic protein-protein interactions via the cadherin repeats
(Tepass, 1999
). Five cadherins
have been isolated in Drosophila:
DE-cadherin/shotgun
(Tepass et al., 1996
;
Uemura et al., 1996
),
DN-cadherin/Cadherin-N
(Iwai et al., 1997
),
flamingo/starry night (fmi; stan FlyBase)
(Usui et al., 1999
;
Chae et al., 1999
),
fat (ft) (Mahoney et
al., 1991
) and dachsous (ds) (Clark et al.), and
several more have been predicted (Hill et
al., 2001
). Cadherins are also involved in cell proliferation
(Mahoney et al., 1991
;
Garoia et al., 2000
) and
tissue organization (Uemura et al.,
1996
; Tepass et al.,
1996
), although the molecular mechanisms underlying their role in
these processes are unclear.
Studies of the PCP pathway in the alignment of hairs within the wing
(Strutt and Strutt, 2002;
Ma et al., 2003
) and the
abdomen (Lawrence et al.,
2002
), and in ommatidial rotation during eye development
(Yang et al., 2002
;
Rawls et al., 2002
), have
shown that Fmi, Ds and Ft, in combination with the transmembrane protein
Four-jointed (Fj) (Villano and Katz,
1995
), establish a gradient of polarity signal within the
epithelium that contributes to the asymmetrical distribution of Fz within the
cell membrane (Adler,
2002
).
The Drosophila Wnt-protein Wingless (Wg) acts as the main ligand
in the canonical Wnt pathway. Binding of Wg to Fz and the co-receptor Arrow
(Wehrli et al., 2000) prevents
the degradation of Armadillo (Arm)/ß-catenin by the APC/Axin/Zw3 complex,
leading to its stabilization and accumulation within the cytoplasm
(Henderson and Fagotto, 2002
).
Once stabilized, Arm protein moves to the nucleus and activates target gene
expression in complex with the transcription factor Pangolin Pan/dTCF
(Brunner et al., 1997
;
Behrens et al., 1996
;
Huber et al., 1996
;
Molenaar et al., 1996
;
Korinek et al., 1997
;
Korinek et al., 1997
;
Kuhl and Wedlich, 1997
), and
with the participation of the nuclear factors Legless (Lgs) and Pygopus (Pygo)
(Belenkaya et al., 2002
;
Kramps et al., 2002
;
Parker et al., 2002
;
Thompson et al., 2002
).
The imaginal disc serves as an excellent model system to gain important
insight into the role of the Wg pathway in pattern formation
(Klein, 2001). During larval
development, two initial groups of 30-50 imaginal cells proliferate and
differentiate to form the adult wings and thorax. Crucial to the growth and
patterning of the wing disc is its subdivision into compartments, a sequential
process that starts early and implicates the differential activation of
selector genes
(Garcia-Bellido et al.,
1973
).
The expression of engrailed (en) and apterous
(ap) in cells of the posterior (P) and dorsal (D) compartments,
respectively, confers distinct adhesion properties to the cells to prevent
them from intermingling with anterior (A) and ventral (V) cells that do not
express those genes (Dahmann and Basler,
1999; Blair, 2001
).
The minimal contact between cells from opposite compartments leads to the
formation of a straight border along the AP and DV interfaces. These
compartment boundaries serve as sources of the signaling molecules
Decapentaplegic (Dpp) and Wg, which coordinate cell proliferation and
patterning in the disc (Tabata,
2001
). At second larval instar, when the wing disc contains only a
few hundred cells, an additional subdivision occurs along its proximodistal
(PD) axis, which segregates cells into notum, hinge and wing
(Klein, 2001
). One of the
earliest signals in this process is the expression of Wg in a group of
anterior cells located at the distal-most part of the disc
(Ng et al., 1996
). This Wg
expression defines the wing territory by the differential activation of
vestigial (vg) within the domain and homothorax
(hth) in the surrounding cells. Within the wing territory, Wg
represses the expression of teashirt (tsh), which promotes
body wall formation (Wu and Cohen,
2002
), and the activity of the Epidermal growth factor receptor
(Egfr) pathway, which specifies notum fate by activating the expression of the
Iroquois complex (iro-C) genes within the proximal-most region of the
disc (Wang et al., 2000
;
Zecca and Struhl, 2002
). The
elimination of this early wg function causes a transformation of the
wing territory into an ectopic notum
(Couso et al., 1993
).
Reported data in vertebrates (Polakis,
2000) and invertebrates
(Sanson et al., 1996
) indicate
that cadherins can modulated Wg signal transduction by affecting the balance
between the levels of cytoplasmic and membrane anchored Arm/ß-catenin
(Vleminckx and Kemler, 1999
).
Here, I describe a new role of the cadherin Ds in pattern formation when
territories along the PD axis are specified in the wing disc. This study
suggests that localized expression of Ds controls PD subdivision by modulating
the response to Wg.
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Materials and methods |
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Clonal analysis and ectopic expression experiments
The FRT/FLP technique (Xu and Rubin,
1993) was used to induce clones of ds mutant cells in
animals of the following genotypes: hsFLP122; FRT40A
dsD36/FRT40A Minute(2L) arm-lacZ and hsFLP122; FRT40A
ds38k/FRT40A Minute (2L) arm-lacZ. Larvae were heat shocked at
36±12 and 60±12 hours after egg laying (AEL). Mutant cells were
marked by the absence of anti-ß-galactosidase antibody staining.
Rescue assays were performed in the following genotypes: omb-Gal4; ds38k/SM5-TM6b X ds38k; UAS-wgts/SM5-TM6b w; ds38k; dppdisk-Gal4/SM5-TM6b X ds38k; UAS-wgts/SM5-TM6b omb-Gal4; ds38k/SM5-TM6b X ds38k; UAS-dpp/SM5-TM6b w; ds38k; dppdisk-Gal4 SM5-TM6b X ds38k; UAS-dpp/SM5-TM6b
Larvae were developed at 25°C except when UAS-wgts was overexpressed; in those cases they were raised at 18°C. At 18°C, the levels of Wg were able to promote ectopic cell proliferation in the hinge without changing the cell fate to wing.
Histochemistry
Imaginal discs were dissected and stained as described previously
(Gomez-Skarmeta et al., 1995).
The following primary antibodies were used: mouse anti-Nub
(Ng et al., 1995
), rat anti-Ci
(Motzny and Holmgren, 1995
),
guinea pig anti-Hth (Azpiazu and Morata,
2000
), rabbit anti-Vg
(Williams et al., 1991
),
rabbit anti-Tsh (Wu and Cohen,
2002
), rat anti-Ds (Yang et
al., 2002
), rat anti-Iro (Diez
del Corral et al., 1999
), rat anti-Zfh2
(Whitworth and Russell, 2003
),
mouse anti-Wg and mouse anti-En (Iowa University Hybridoma Bank), rabbit
anti-ß-galactosidase (Cappel) and mouse anti-ß-galactosidase
(Amersham). Fluorescent secondary antibodies were from the Jackson
Immunostaining Laboratory.
Adult cuticles
For microscopic examination, the wing and legs were dissected and treated
in 10% KOH and mounted in a solution of lactic acid mixed 6:5 with
ethanol.
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Results |
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From an allelic series that includes most of the ds alleles,
ds38k represents the strongest hypomorphic condition. In
addition to the classical phenotype, a percentage of
ds38k escapers (approximately 5%) show striking phenotypes
that were not described previously. These consist of the presence of a lateral
protuberance similar to an ectopic scutum (sc) and scutellum (sct), indicating
a possible notum duplication (Fig.
1A,B), and the replacement of the normal wing
(Fig. 1C) by a winglet
(arrowhead in Fig. 1A,B). The
ectopic notum and the winglet are always associated. A comparison with the
wild-type wing (Fig. 1C,D)
shows that the winglet is composed of proximal anterior structures that are
arranged in a mirror-image duplication
(Fig. 1E). The smallest winglet
is exclusively formed by a duplication of the tegula and humeral sclerite
structures (Fig. 1B,
arrowhead), whereas the largest one also has a rudimentary wing blade composed
of a small costa and anterior wing margin
(Fig. 1E,F). I shall refer to
these newly described anomalies as the double-notum-winglet
(DNW) phenotype. This phenotype is also found in Df(2L)S2 homozygous
flies (1 out of 100 heminota), and in individuals from heterozygous
combinations of ds38k and other strong alleles, such as
ds33k (3 out of 72 heminota), the P insertion
ds-lacZ (approximately 2% of heminota) and
dsUA071 (Adler et al.,
1998).
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Two other striking features of ds mutant discs are the relative
position of the prospective wing pouch with respect to the AP border, and the
differences in size between the A and P compartments, as revealed by the
expression of Cubitus interruptus (Ci)
(Fig. 1G, part b; I, red) and
En (Fig. 1G, part c; J, green),
respectively. In the wild type, the wing pouch is subdivided in two
compartments of a similar size (Fig.
1G, parts b,c). By contrast, the reduced wing pouch in DNW discs
is located entirely within the A compartment
(Fig. 1G, part c; J,K). These
observations are in agreement with the exclusive presence of anterior
structures in the ds38k winglet
(Fig. 1E,F). Nevertheless, the
A and P compartments are both present within the notum, where they are of a
normal size (Fig. 1G, part b;
I). Note that the extant and the ectopic nota
[Fig. 1H,I (n,n')] are
arranged in a mirror-image disposition, and that the Iro domains are kept in
contact (Fig. 1H, dotted line),
in contrast to wg mutant discs in which they are separated by a wide
stripe of hinge cells (Cavodeassi et al.,
2002). The absence of P cells in the wing territory was observed
as early as the mid second instar (Fig.
1J, inset). However, the size of the D and V compartments do not
seem to be altered, as revealed by the expression of Ap in dorsal cells
(Fig. 1G, part d; K, red).
ds is expressed in the wing disc from early stages of development
To gain insight into the role of ds during early wing development,
I examined the expression of ds in the wing disc of second and early
third instar larvae. ds expression was monitored by the
ds-lacZ reporter gene, which reflects the spatial pattern of
ds mRNA (Clark et al.,
1995). In second instar larvae, ds-lacZ expression is
essentially confined to the distal part of the wing disc
(Fig. 2A,B), but is absent in
those distal A cells in which Wg strongly accumulates
(Fig. 3A, green). This Wg
expression constitutes the earliest marker for the nascent wing pouch
(Couso et al., 1993
;
Ng et al., 1996
). Soon
thereafter, when Wg expression is expanded to the adjacent P cells,
ds-lacZ expression fades away
(Fig. 2C) and becomes confined
to a ring of cells around the prospective wing pouch
(Fig. 2D). At this stage, most
of the hinge cells located between the prospective notum and wing pouch
express ds-lacZ at high levels, as revealed by the Iro and Nub
markers (Fig. 2D). A weak
expression of ds-lacZ overlaps with the periphery of the Nub domain
(Fig. 2D) and marks the region
that will become the proximal wing. At third instar, ds-lacZ
expression is also observed within the lateral regions of the prospective
notum (Fig. 2F). An antibody
directed against the cytoplasmic region of Ds protein
(Yang et al., 2002
) reveals a
Ds protein distribution similar to the ds-lacZ expression pattern and
an apical location at the plasma membrane
(Fig. 2E). From these results,
I conclude that ds-lacZ expression is one of the earliest and most
specific markers of the prospective hinge during the second and early third
larval instar.
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In the wild type, the initial wing territory is specified around second
instar by the expression of Wg in distal A cells. ds-lacZ is almost
eliminated within the nascent wing primordium, except in a few cells at the
periphery, which express both wg and ds-lacZ at low levels
(Fig. 3A). A similar expression
pattern was also observed for hth and zfh2
(Casares and Mann, 2000;
Whitworth and Russell, 2003
).
By contrast, tsh expression is turned off within the Wg domain, and
in a ring of surrounding cells, but remains uniformly expressed in the rest of
the wing disc (Fig. 3B) (Wu and Cohen, 2002
). The
pattern of ds-lacZ expression with respect to Hth, Tsh and Zfh2 is
maintained until the third instar (Fig.
3C,D,E). To visualize the distal border of each expression domain
in more detail, I also examined optical cross-sections of late third instar
wing discs. ds-lacZ, zfh2 and hth are co-expressed at high
levels in a ring of cells abutting the Nub domain
(Fig. 3C,E,F; arrow in F). Tsh
is repressed within these cells (Fig.
3D) by the activity of Wg (Fig.
3D,G; arrow in G) (Wu and
Cohen, 2002
). Based on these expression patterns, I can define
three concentric domains with respect to Nub expression
(Fig. 3N). The cells of the
innermost ring (ring I) express nub and low levels of
ds-lacZ and hth (Fig.
3F, folds 1 to 3), and will give rise to the proximal wing
structures eliminated in wgspd-fg
(Neumann and Cohen, 1996
) and
zfh2MS209 (Whitworth
and Russell, 2003
) mutants. The middle ring (ring II) spans the
region in which tsh is repressed
(Fig. 3G, folds 2 to 4), and in
which ds-lacZ and hth are expressed at high levels
(Fig. 3F, folds 2 to 4). These
cells will develop the hinge structures eliminated in
ds38k flies. Finally, the outermost ring (ring III)
consists of cells that co-express low levels of ds-lacZ and
hth within the Tsh domain (Fig.
3G, folds 4 to 5). Those cells will form the body wall structures
excluded from the notum territory, such as the thoracic pleura.
DNW discs from early larval stages suggest altered specification of the proximal wing and hinge territories (Fig. 1J, inset). Two main differences are observed in DNW discs with respect to the wild-type discs. First, hth and ds-lacZ expression is eliminated from rings I and III. Only a few cells in the ring II are still expressing both genes (Figs 3J,K). Second, cells within ring III maintain Tsh expression (Fig. 3L), but now express notum-specific genes such as iro-C (Fig. 1H). Note that the zfh2 domain is wider than the domains of hth and ds-lacZ expression (Fig. 3M). Thus, reduction of hth and zfh2 expression might be the cause of the loss of proximal wing and hinge territories in DNW discs. The ectopic notum observed in DNW flies is most likely to derive from the outermost ring of cells that missexpress the iro-C genes.
Ds regulates Wg signaling
The notum duplication observed in DNW flies resembles a similar phenotype
described for wg1 flies
(Sharma and Chopra, 1976). In
general, a decrease of Wg signaling during early wing disc development
transforms the wing into an ectopic notum
(Brunner et al., 1997
; Kramps,
2002). However, DNW flies retain a rudimentary wing, suggesting that the
ectopic notum in these mutants is formed predominantly at the expense of
proximal wing and hinge structures.
Wg signaling has been shown to control both hth and zfh2
expression during the specification of these structures
(Casares and Mann, 2000;
Whitworth and Russell, 2003
).
The analysis of DNW discs described above indicates that these same genes are
affected by the reduction of Ds activity, suggesting a role of Ds in Wg
signaling. In order to investigate this possibility, I tested whether an
increase of Wg protein levels could rescue the DNW phenotype of ds
mutant discs. An ectopic dose of Wg was provided to
ds38k/ds38k discs using the UAS-wg
transgene in combination with either the dpp-Gal4 or
omb-Gal4 drivers. Under these conditions, the DNW discs of
ds38k larvae were completely rescued in size and pattern
(Fig. 4A) (over 130 larvae were
analyzed). The rescued wing discs consisted of just a single notum and a wing
pouch of normal size and AP subdivision. In particular, the specification of
the proximal wing and hinge territories was restored, as assessed by
hth expression (Fig.
4B; although the hinge domain is expanded compared with the wild
type), due to the later role of Wg in the induction of cell proliferation
within the hinge (Neumann and Cohen,
1996
). The ds38k larvae expressing
UAS-wg under dpp-Gal4 control also showed enhanced
viability, although they died before reaching adulthood. Thus, these data
suggest that all aspects of the DNW phenotype are caused by a reduction in Wg
signaling.
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In order to test whether other signaling pathways are affected by the
reduction of Ds activity, I ectopically expressed Dpp in ds mutant
discs using a similar approach to that described for ectopic wg
expression. Dpp secreted by a thin stripe of cells along the AP boundary acts
directly, and at long range, on all cells within the developing wing pouch to
organize pattern formation and growth
(Capdevila et al., 1994;
Zecca et al., 1995
;
Burke and Basler, 1996
;
Lecuit et al., 1996
;
Nellen et al., 1996
). In
addition, Dpp also functions outside of the wing pouch to confine Iro-C
expression to the notum (Cavodeassi et
al., 2002
). In wild-type discs, the Dpp source (AP border) is
located at the center of the prospective wing pouch
(Fig. 1G, part a; white line).
As the AP border is far removed from the wing pouch in DNW discs
(Fig. 1J,K), a reduced level of
Dpp within the wing pouch cells could be responsible for the winglet
phenotype. To address this issue, UAS-dpp was expressed under
dpp-Gal4 control in ds38k larvae. Under these
experimental conditions, the DNW phenotype was retained. By contrast,
ds38k larvae bearing the UAS-dpp and
omb-Gal4 transgenes produced wing discs with a remarkable expansion
of the wing pouch (Fig. 4C,D).
The rescued wing pouch is formed of A and P cells, indicating that Dpp also
contributes to the recruitment of P cells into the wing fate
(Fig. 4D). However, the notum
duplication was still present in these discs
(Fig. 4C,D; insets). In
summary, I conclude that a disruption of Wg signaling is mainly responsible
for the hinge to notum transformation in DNW flies. However, the winglet
phenotype due to the absence of P cells recruited into the wing fate is
caused, not only by the lack of Wg, but also by Dpp expression, although the
DNW phenotype retained in the UAS-dpp/dpp-Gal4 combination suggests
that only cells specified into the wing fate by Wg expression can respond to
Dpp in this process. Finally, the data indicate that Ds is required for
efficient Wg and/or Dpp signaling for the recruitment of the P cells into the
wing fate during early stages of wing development.
PD patterning requires Ds in Wg-receiving cells during early stages of disc development
The analysis of ds mutant discs reveals early patterning defects
(Fig. 1J, inset). To assess the
temporal requirement of Ds in Wg-mediated PD patterning, I analysed
zfh2 expression (Fig.
5A, inset) in ds mutant clones. zfh2 is
activated by Wg in second instar wing discs to become independent soon
thereafter (Whitworth and Russell,
2003). Using the Minute technique
(Morata and Ripoll, 1975
), I
generated large clones of dsD36cells at different stages
of larval development. Clones induced at early second instar, eliminate
zfh2 expression (Fig.
5A,B). Moreover, an overgrowth was observed within the mutant
territory, similar to that described for mutations in other cadherins, such as
ft (Mahoney et al.,
1991
). By contrast, clones of dsD36 cells
induced later do not eliminate zfh2 expression, although expression
levels were reduced in the hinge cells
(Fig. 5C,D; asterisk). Thus, it
seems that Ds, similar to Wg signaling, is essential for the initiation, but
not the maintenance, of zfh2 expression at later developmental
stages. Together, these findings support a role for Ds in Wg-mediated
initiation of zfh2 expression during early stages of wing
development. I have previously shown that wg and ds are
expressed in complementary domains at these stages
(Fig. 3A), thus I conclude that
Ds must act in Wg-receiving cells to achieve PD patterning.
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ds regulates Wg signaling in other developmental contexts
I also investigated whether Ds is required for Wg-mediated patterning in
imaginal discs other than the wing disc by analyzing the genetic interactions
between ds and several components of the Wg pathway during leg
development. Homo- and hetero-allelic combinations of ds cause a
reduction of the segment size and fusion of the tarsal segments, with partial
elimination of the tarsal joints (Fig.
7B,D) (Clark et al.,
1995). This phenotype resembles some defects associated with the
loss of function of pangolin/dTCF,
(Brunner et al., 1997
) and
legless/BCL9 (Kramps et al.,
2002
). Therefore, the levels of Wg signaling were manipulated in
mid-strength heteroallelic combinations of ds. The loss of one
wild-type copy of dsh enhanced the fusion of leg tarsi and shortened
the leg segments (Fig. 7B,C).
By contrast, the leg phenotype of ds showed a complete recovery of
the tarsal joints and an increase in the length of the segments when one dose
of the nkd gene, an antagonist of the Wg pathway, was eliminated
(Zeng et al., 2000
;
Rousset et al., 2001
)
(Fig. 7D,E). Taken together,
these findings support a more general role for ds in Wg-mediated
patterning processes.
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Discussion |
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ds is required for early specification of the proximal wing and hinge
The wing primordium is specified as a few anterior cells that express
wg at the distal-most part of the wing imaginal disc at second larval
instar. Slightly later, wg is also expressed in P cells and these
cells are recruited into the wing fate (Ng
et al., 1996). In DNW discs, the level of Ds protein is highly
reduced and only the initial anterior group of Wg-expressing cells becomes
specified into the wing fate (Fig.
1H,J). The levels of this initial Wg expression seems not to be
affected in DNW discs. However, neither the P cells abutting the initial
anterior Wg domain nor the surrounding cells of this early wing primordium are
able to respond to Wg, leading to the formation of a wing pouch composed
exclusively by A cells (Fig.
1I,J). Moreover, the activation of Wg target genes, such as
hth, required for the specification of hinge cells fails in DNW discs
(Fig. 3J), and, consequently,
the proximal wing and hinge structures do not develop
(Fig. 1B,E)
(Casares and Mann, 2000
;
Whitworth and Russell, 2003
).
The significantly reduced rings of ds-lacZ, hth and zfh2
expression (Fig. 3J,K,M) in DNW
discs most likely reflect the residual Ds activity retained in the
ds38k mutant. Cells close to the Wg source might thus
still be able to respond to high Wg levels during early stages of wing
development. However, under null conditions for ds
(dsD36) the expression of zfh2 is eliminated
(Fig. 5A,B).
Thus, in addition to its function in PCP, ds plays a role in early
patterning when the specification of the different territories along the PD
axis takes place in response to Wg. Initially, ds facilitates the
recruitment of P cells into the wing fate in response to Wg. Subsequently, Ds
promotes the activation of Wg target genes in the surrounding cells to specify
the hinge. Note that once the hinge cells have been specified in response to
Wg signaling, ds seems to be dispensable for global wing disc
patterning, as the classical ds38k phenotype
shows (Clark et al., 1995). In
this case, only mild defects such as slight tissue overgrowth or polarity
defects were observed, suggesting additional functions of ds related
to cell adhesion.
As shown above, ectopic expression of Dpp (Fig. 4D) in wing cells of DNW discs restores both the formation of the AP border and cell proliferation within the wing pouch, indicating that both Wg and Dpp orchestrate these events. Only cells previously committed to the wing fate by Wg are able to proliferate in response to Dpp, as the UAS-dpp/dpp-Gal4 and UAS-dpp/omb-Gal4 experiments suggest. In the ds mutant background, omb is expressed in anterior wing cells, albeit in the absence of the AP border/Dpp source within the wing pouch, suggesting that this initial omb expression might not be Dpp dependent. Similar results were observed for spalt (sal), another known target gene of dpp. I propose that Ds primarily regulates Wg signaling in the initial recruitment of P cells into putative wing territory. Once this initial recruitment has occurred, Dpp expression is established and Dpp signaling can contribute to the further recruitment of P cells. Expression of UAS-dpp in anterior wing pouch cells of ds mutant discs using omb-Gal4 can bypass the initial requirements for Wg in P cell recruitment, leading to the observed wing pouch rescue (Fig. 4C,D).
Ds contributes to the maintenance of the hinge/notum boundary
In vertebrates, during telencephalon formation, the organization into
different structures requires the expression of different cadherins in
adjacent regions to maintain a compartment boundary based on differential cell
affinity features. It has been suggested that the expression pattern of each
of these cadherins is under the control of specific signaling cascades
(Inoue et al., 2001).
In Drosophila, during imaginal disc development, indirect evidence
has suggested that cell adhesion might be under the control of the same
signaling pathways that control cell proliferation and patterning. The smooth
borders of clones mutant for thick vein (tkv), the receptor
of Dpp (Burke and Basler,
1996), or smoothened (smo)
(Blair and Ralston, 1997
;
Rodriguez and Basler, 1997
), a
downstream component of the Hedgehog (Hh) signaling pathway, indicate that
mutant cells change their affinity properties and therefore try to minimise
the contact with surrounding wild-type cells. Nevertheless, little is known
about the molecules involved in these adhesiveness differences. Recent work
has proposed that both tartan and capricious
(caps), two transmembrane proteins regulated by ap, are
putative candidates to maintain the affinity boundary between dorsal and
ventral cells (Milan et al.,
2001
). However, whereas clones ectopically expressing
tartan and caps in V cells tend to contact D cells, the
elimination of tartan and caps in clones from D cells had no
effect on DV boundary formation.
In the DNW phenotype, the ectopic notum develops from cells of the hinge
territory (Fig. 1A,B).
According to the proposed subdivision into concentric rings (I to III), cells
from the outermost ring III expressing Tsh and Ds will give rise to that part
of the body wall that is excluded from the notum region
(Fig. 3N, wt). In DNW discs,
the absence of Ds produces an expansion of notal-specific iro-C
expression to more distal positions to fill up the Tsh domain (compare
Fig. 1H with
Fig. 3L,N;
ds/). These distal cells acquire a notum fate
(Fig. 3N), generating an
ectopic notum similar to wg1 mutant flies
(Sharma and Chopra, 1976).
Thus, Ds protein contributes to hinge/notum boundary formation by means of an affinity border. This process would occur at early second instar when Iro-C expression is capable of specifying the notum fate. This finding provides the first evidence that a cadherin is able to maintain the cell boundary between two adjacent territories in Drosophila.
How does ds participate in Wg signaling?
Several findings point out a specific role of Ds in the modulation of Wg
signaling: (1) the elimination of zfh2 expression in ds
mutant clones (Fig. 5A,B); (2)
the genetic interactions of ds alleles with several components of the
Wg signaling pathway; and (3) the rescue of the DNW phenotype by increasing Wg
levels. It has been shown that Ds is associated with adherens junctions at the
apical surface of the imaginal cells (Fig.
2E) (Ma et al.,
2003), to mediate cell-cell adhesion. A major step of the cell
adhesion mechanism requires interaction of the cytoplasmic tail with
Arm/ß-catenin to connect the cadherin-catenin complex to the actin
cytoskeleton (Vleminckx and Kemler,
1999
). Thus, the phenotype could reflect changes in the balance
between cytoplasmic Arm versus Arm anchored to the plasma membrane. If this
were the case, then a reduction of ds function would increase Wg
signaling; however, the results presented above indicate that loss of
ds decreases Wg signaling. Moreover, sequence analysis has shown that
the ß-catenin binding motifs in the Ds protein, which have to be in
tandem to be functional, are separated by a stretch of amino acids, further
discarding the possibility that Ds binds directly to Arm to modulate its
cytoplasmic levels (Clark et al.,
1995
).
Alternatively, the apical plasma membrane acts as a structural centre that
contains crucial components that modulate the Wg pathway, such as Dsh
(Cliffe et al., 2003;
Axelrod, 2001
), E-APC
(Yu et al., 1999
) and Axin
(Cliffe et al., 2003
). Axin
and E-APC, promote the degradation of cytoplasmic Arm, the main effector of
the Wg cascade (Ikeda et al.,
1998
; Yu et al.,
1999
). Previous work has shown that, upon binding of Wg in the
receiving cells, the Axin/E-APC complex becomes anchored to the plasma
membrane to prevent Arm degradation
(Kishida et al., 1998
;
Cliffe et al., 2003
). In this
context, Ds protein, as part of the adherens junctions, could be the cadherin
required to anchor this degradation complex to the plasma membrane. In
ds mutant cells, the cytoplasmic levels of the Axin/E-APC complex
would be higher and, therefore, Wg signaling would decrease. In agreement with
this hypothesis, I have observed that mild ds phenotypes are enhanced
when a copy of dsh gene is eliminated
(Fig. 7C). Still, Ds could act
at the level of Wg reception, by increasing the Fz/Wg-binding affinity or by
recruiting Fz molecules to the apical plasma membrane, as has been
demonstrated for the cadherin Fmi in the PCP processes
(Strutt, 2001
).
Early anterior Wg activity initiates specification of the PD axis in the wing disc
To date, the current model explaining the specification of the territories
along the PD axis assumes that the initial anterior Wg expression at second
instar is required only for cells to acquire the wing fate. It is only later,
when wg is expressed in two concentric rings that its function is
required to specify the hinge territory.
Wg has been shown to be required for the development of the hinge. On the
one hand, Wg activates downstream genes such as hth
(Casares and Mann, 2000) or
zfh2 (Whitworth and Russell,
2003
) to specify the hinge fate. On the other hand, Wg controls
cell proliferation when it is expressed from early third instar into the IR
and OR rings (Neumann and Cohen,
1996
; Del Alamo et al.,
2002
). It has been established that the specification of the hinge
takes place later than the wing; however, my data show that an early and
timely limited depletion of Wg activity causes a failure in hinge
specification. This is mainly based on the observation that only early-induced
ds clones abolish zfh2 expression required for hinge
formation. In ds mutant clones induced later, hinge development is
unaffected, although a perdurance of ds activity in these clones
cannot be excluded. Still, the rescue of hinge development in DNW discs that
ectopically express Wg under dpp-Gal4 further support an early
specification of the hinge. In these discs, ectopic Wg expression stays
confined to the AP border. At early stages, the AP border must be located
close enough to the nascent wing primordial to allow the spreading of Wg into
regions destined to become hinge territory. At late stages, the narrow stripe
of ectopic Wg expression can no longer account for the maintenance of the
whole hinge territory. It is rather the Wg within the IR and OR that maintains
hth expression and, with it, the specification of the hinge fate. At
this stage, either Wg works independently of ds or its requirements
for ds are lower. Thus, if hinge specification is not initiated early
upon ds and wg activities, wg expression cannot be
established and the development of the hinge is aborted.
The present results provide insights that help us to understand how the PD axis is established in the wing disc. The initial event in this process would be the early activity of Wg. When Wg is expressed at the distal part of the wing disc in a small group of anterior cells, it not only promotes the activation of target genes like vg, nub or scalloped (sd) in the wing cells, but also the expression of hth and zfh2 to specify the hinge. At the same time, Wg would repress tsh or vein (vn) at the distal part of the wing disc to separate the proximal wing and hinge regions from the body wall where Egfr signaling activates notum-specific genes like iro-C. Thus, in cooperation with dpp, wg establishes the AP and PD axis in the prospective wing and hinge regions.
In DNW discs, even though the Dpp source is distantly and asymmetrically located with respect to the wing pouch (Fig. 1G, part c; J), anterior wing cells differentiate into distinct cell types (Fig. 1E,F) in a mirror image disposition. This result suggests that specific positional information might be provided independently of dpp. Ap in combination with Wg might contribute to this initial AP positional information. Once P cells are recruited into the wing fate, Dpp takes over and promotes pattern formation along the AP axis, as well as proliferation within the wing pouch.
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ACKNOWLEDGMENTS |
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