Glypicans shunt the Wingless signal between local signalling and further transport
Xavier Franch-Marro,
Oriane Marchand,
Eugenia Piddini,
Sara Ricardo,
Cyrille Alexandre and
Jean-Paul Vincent*
National Institute for Medical Research, The Ridgeway Mill Hill, London
NW7 1AA, UK
*
Author for correspondence (e-mail:
jp.vincent{at}nimr.mrc.ac.uk)
Accepted 23 November 2004
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SUMMARY
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The two glypicans Dally and Dally-like have been implicated in modulating
the activity of Wingless, a member of the Wnt family of secreted glycoprotein.
So far, the lack of null mutants has prevented a rigorous assessment of their
roles. We have created a small deletion in the two loci. Our analysis of
single and double mutant embryos suggests that both glypicans participate in
normal Wingless function, although embryos lacking maternal and zygotic
activity of both genes are still capable of transducing the signal from
overexpressed Wingless. Genetic analysis of dally-like in wing
imaginal discs leads us to a model whereby, at the surface of any given cell
of the epithelium, Dally-like captures Wingless but instead of presenting it
to signalling receptors expressed in this cell, it passes it on to
neighbouring cells, either for paracrine signalling or for further transport.
In the absence of dally-like, short-range signalling is increased at
the expense of long-range signalling (reported by the expression of the target
gene distalless) while the reverse is caused by Dally-like
overexpression. Thus, Dally-like act as a gatekeeper, ensuring the sharing of
Wingless among cells along the dorsoventral axis. Our analysis suggests that
the other glypican, Dally, could act as a classical co-receptor.
Key words: Wingless, Glypicans, Morphogen transport, Drosophila, Signalling
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Introduction
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Extracellular ligands are typically degraded after engagement with their
cognate receptors and activation of the signalling pathway. Degradation is
usually initiated by receptor-mediated endocytosis. This raises a fundamental
problem for ligands that must act over long distances within epithelia.
High-affinity signalling receptors are expected to trap the ligand near the
source and thus prevent transport to more distant cells
(Chen and Struhl, 1996
;
Kerszberg and Wolpert, 1998
).
How is a locally produced ligand appropriately allocated to all the cells
within a field, near and far? One possibility is that specialised,
non-signalling receptors ensure long-range signalling. In wing imaginal discs
of Drosophila, the secreted glycoprotein encoded by wingless
is produced at the prospective wing margin and spread symmetrically along the
dorsoventral axis (Zecca et al.,
1996
; Cadigan et al.,
1998
). One class of receptors that could modulate the distribution
of Wingless are heparan sulphate proteoglycans (HSPGs). In particular,
overexpression of the glypican Dally-like (Dlp) causes accumulation of
Wingless at the surface of imaginal disc cells
(Baeg et al., 2001
). Somehow,
such accumulation is accompanied by a reduction in Wingless signalling.
Although this gain-of-function experiment suggests that Dally-like could
regulate the distribution of Wingless, so far, this has not been confirmed by
loss-of-function analysis. Indeed, the normal role of Dlp in Wingless function
is still unknown.
Good evidence for the involvement of HSPGs in Wingless function comes from
the isolation and analysis of mutants in genes encoding HSPG modifying
enzymes. Drosophila embryos lacking sugarless, which encodes
UDP-Glucose-Dehydrogenase, an enzyme required for heparan sulphate
biosynthesis, are deficient in Wingless signalling
(Binari et al., 1997
;
Hacker et al., 1997
;
Haerry et al., 1997
).
Likewise, embryos lacking sulfateless, which encodes heparan sulphate
N-deacetylase/N-sulphotransferase (an enzyme needed for the modification of
heparan sulphate) also resemble wingless mutants
(Lin and Perrimon, 1999
).
Further genetic analysis showed that HSPG metabolising enzymes are also
implicated in additional signalling pathways such as those activated by
Hedgehog, FGF and Dpp (Bellaiche et al.,
1998
; Lin et al.,
1999
; Han et al.,
2004a
; Takei et al.,
2004
). To what extent HSPGs play distinct roles in the different
signalling pathways remains to be determined. Practically, the involvement of
HSPGs in both Wingless and Hedgehog signalling complicates genetic analysis in
embryos because the terminal phenotype of wingless and
hedgehog mutants look very similar to each other, making it difficult
to distinguish roles in either or both signalling pathways. In imaginal discs,
Wingless and Hedgehog signalling are easily distinguished experimentally and,
in this tissue, it is clear that loss of sulfateless causes a
reduction in Wingless signalling (Baeg et
al., 2001
). In particular, Wingless protein that normally
accumulates at the surface of wild type Wingless-expressing cells does not do
so at the surface of sulfateless mutant cells, suggesting a potential
role of HSPGs in tethering Wingless at the cell surface.
Genetic analysis of HSPG modifying enzymes clearly implicates HSPGs in
Wingless function. What are the relevant HSPGs involved? Four HSPGs are
predicted to be encoded by the Drosophila genome
(Nybakken and Perrimon, 2002
).
Of these, as mentioned above, Dlp is a good candidate on the basis of
gain-of-function studies. However, RNAi injection in embryos has failed to
confirm a role for Dlp in Wingless signalling. Instead, a clear role in
Hedgehog signalling was demonstrated by RNAi
(Desbordes and Sanson, 2003
;
Lum et al., 2003
) and this was
later confirmed with traditional genetic mutants
(Han et al., 2004b
). In these
experiments, a possible role of Dally-like in Wingless signalling might have
been masked by redundant activity from another glypican. Indeed, the other
glypican found in Drosophila, Dally, has been implicated in Wingless function
on the basis of RNAi-induced phenotypes in embryos
(Lin and Perrimon, 1999
)
although this has been questioned by subsequent experiments
(Desbordes and Sanson, 2003
).
Nevertheless, weak wingless-like phenotypes are seen in presumed weak
allele of dally (Lin and
Perrimon, 1999
; Tsuda et al.,
1999
).
Overall, various experiments involving presumed weak alleles, RNAi
injection or overexpression have implicated Dally and Dlp in Wingless function
(Nakato et al., 1995
;
Lin and Perrimon, 1999
;
Tsuda et al., 1999
;
Baeg et al., 2001
). However, a
clear assessment of their role has been hampered by the involvement of HSPGs
in other signalling pathways and also by the lack of suitable null mutants
(Sanson, 2004
). We have
created null mutants in both dally and dlp. The analysis of
single and double mutant embryos shows that both glypicans participate in
normal Wingless function. Using a combination of gain- and loss-of-function
approaches in imaginal discs, we have investigated the specific roles of the
two glypicans in regulating the activity and distribution of the Wingless
signal in wing imaginal discs.
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Materials and methods
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DNA constructs
To create an N-terminal FLAG tagged version of Dally, a double-stranded
oligonucleotide containing three copies of the FLAG epitope was inserted in
the unique AatII site (position 150 in Dally). The Dally-FLAG cDNA
was subsequently inserted in pUAST as an EcoRI-XbaI
fragment. DNA carrying UAS-Dally-HA and UAS-Dlp-HA as well as pMT-Gal4 used
for the binding experiments with S2 cells were obtained from S. Cohen, EMBL.
See Fig. 1B for a diagram of
the constructs.

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Fig. 1. Molecular lesions in dlpMH20 and
dallyMH32 and organisation of the tagged glypicans used.
(A) Diagram of the genomic regions of dlp and dally. The
position of the original P-elements is shown with an inverted triangle. The
deletions in dlpMH20 and dallyMH32 are
indicated as small black bars (to scale). The exact break points relative to
the predicted start of transcription are -42 to +467 for dlp (ATG is
at 454) and -472 to +1410 for dally (ATG is at 728). The deficiencies
used in complementation assays uncover the whole region with break points
located far away and are represented as long black lines [Df(3L) ED4413
dally was generated for this study while Df(3L) ED4543 dlp was
obtained from Drosdel]. (B) The tagged glypicans used in this study. The
location and nature of the tag is shown. HA-Dally was only expressed in
cultured cells while Flag-Dally and Dlp-HA were expressed in transgenic flies
and cells.
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Drosophila stocks
The following transgenic stocks were used for exogenous expression:
UAS-Dally-FLAG (generated for this study), UAS-Dally-like-HA (from S. Cohen,
EMBL). Both were driven with dpp-Gal4, engrailed-Gal4 or
tubulin-Gal4. For loss-of-function studies, we generated mutants in
dally and dlp by imprecise excision of a P-element inserted
in the 5' region (see Fig.
1A). For dlp, we obtained a deletion of nearly 500
nucleotides, which removes the 5'UTR and 16 nucleotides downstream of
the ATG. For dally, we obtained a deletion of about 1.8 kb that
removes the first exon. Both mutants are expected to produce no functional
protein. Clones of mutant cells were induced by Flp-mediated mitotic
recombination. The following additional stocks were used: GSV6 9608 and GSV6
11297 (P-insertions in dlp and dally, respectively, obtained
from Toshiro Aigaki, Tokyo University); Df(3L) ED4413, a large
deficiency that uncovers dally (generated by us with the Drosdel kit,
www.drosdel.org.uk);
and Df(3L) ED4543 (obtained from Drosdel), which uncovers dlp.
w; FRT2A dlpMH20, w; FRT2A
dallyMH32 and w; FRT2A
dallyMH32dlpMH20 were generated for this study.
We also used yw hsflp; FRT2A ubi-GFP (from the Bloomington
Stock Center), wgCX4 and hhAC
(described at
flybase.bio.indiana.edu),
UAS-GFP-Wingless, and sim-gal4 (from Benny Shilo, Weizmann
Institute). Germline clones were induced with yw; FRT2A
ovoD (from the Bloomington Stock Center). To induce
overexpression clones, the following stocks were used: yw hsflp;
tubulin>>GAL4>>UAS lacZ, yw; FRT42D pwn and yw hsflp;
FRT42D Gal-80; tubulin-Gal-4 (from S. Cohen, EMBL).
Embryo preparations
To visualise cuticle patterns, 24-hour-old embryos were mounted in Hoyer's
and photographed under DIC or phase-contrast microscopy. In situ hybridisation
was performed on fixed embryos hybridised with a digoxigenin labelled
rhomboid probe (a gift from J. F. de Celis, Cambridge
University).
Antibodies
Primary antibodies used were mouse anti-Wingless 4D4 (prepared from cells
obtained from the DSHB), mouse M2 anti-Flag (Sigma; 1/1000), mouse anti-HA 1.1
(Covance; 1/500), Alexa-488 labelled mouse anti-HA 1.1 (Covance; 1/500),
rabbit anti-GFP (Abcam; 1/2500), mouse anti Distal-less (from I. Duncan,
University of Wisconsin; 1/500) and guinea pig anti-Senseless (from Hugo
Bellen, Baylor College of Medicine; 1/1000). Secondary antibodies (all from
Molecular Probes) were Alexa488-conjugated goat anti-rabbit (1/200),
Alexa488-conjugated goat anti-mouse (1/200), Alexa594-conjugated goat
anti-mouse (1/200) and Alexa488-conjugated goat anti-guinea pig (1/200).
Wingless-binding assay
GFP-Wingless medium prepared from a stable line (S2-GFP-Wg) was applied to
S2 cells grown on coverslips and transiently transfected with dally
or dlp constructs (HA-tagged) and induced overnight with 0.25 mM
CuSO4. GFP-Wingless binding was performed on ice as previously
described (Bhanot et al.,
1996
). After fixation, GFP-Wingless was detected with an anti-GFP
antibody and the HA tagged receptors with anti-HA.
 |
Results and discussion
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Dally and Dlp contribute to normal Wingless activity in embryos
In order to assess the function of Dally and Dlp, we have created, for each
gene, a small deletion that removes the translation start and the signal
peptide. These alleles are henceforth referred to as
dallyMH32 and dlpMH20 (see molecular
description in Fig. 1). For
both alleles, even if an opportunistic translation start were used, no signal
peptide would be encoded. For dlp, the next possible ATG is 123
residues downstream of the normal translation start, while for dally
the mutation removes the first exon thus deleting at least the first 90
residues. Therefore, in both cases, any protein product would not be allowed
to enter the secretory pathway and reach the cell surface even if a downstream
translation start is used (i.e. no functional protein could be produced). On
the basis of this evidence, we consider dlpMH20 and
dallyMH32 to be null. Previous dally alleles,
including the strongest one, dallyP2 were thought to be
hypomorphic. However, we find that the penetrance and expressivity of adult
phenotypes is similar in dallyMH32 homozygotes as in
dallyP2 homozygotes (as well as in
dallyMH32/Df(3L) ED4413 animals). It appears therefore
that dallyP2 is also a null allele.
As a first assessment of the role of these two proteoglycans in Wingless
function, we analysed embryos that lack the maternal and zygotic contribution
of either dally, dlp, or both. Embryos lacking dally appear
normal, suggesting that this gene is not essential for embryonic development
(data not shown) (see also Desbordes and
Sanson, 2003
). By contrast, and as previously shown, Dlp-deficient
embryos die with a so-called denticle lawn phenotype, which has been
attributed to a failure in transduction of the Hedgehog signal [early targets
of Hedgehog signalling fail to be activated
(Desbordes and Sanson, 2003
;
Lum et al., 2003
)]. Assessing
a possible, additional role in Wingless signalling has been difficult because
the terminal phenotype of embryos lacking Hedgehog signalling is similar to
that of wingless-deficient embryos. To investigate this, we used an
epidermal molecular marker that differentiates between hedgehog,
wingless and wingless hedgehog mutant embryos. As can be seen in
Fig. 2A-C, the pattern of
rhomboid expression is distinct in all three genetic conditions (see
also Alexandre et al., 1999
).
In embryos lacking maternal and zygotic dlp, the pattern of
rhomboid expression is variable with features of both
hedgehog and wingless mutants, but falling short of
phenocopying wingless hedgehog double mutants (not shown). This
suggests a loss of Wingless activity, although an incomplete one, perhaps
because of a redundant contribution from Dally. We therefore assessed the
pattern of rhomboid expression in embryos lacking maternal and
zygotic dally and dlp (dally dlp double mutants for
short). As shown in Fig. 2D, it
strongly resembles that seen in wingless hedgehog double mutants.
This similarity is confirmed by close inspection of terminal cuticular
phenotypes. The cuticle phenotype of dally dlp double mutants is more
severe than that of embryos lacking maternal and zygotic dlp (not
shown) and, importantly, it is characterised by a feature seen in wingless
hedgehog double mutants, the presence of mid-ventral denticles whorls
(Fig. 2E,F). We conclude that
both glypicans participate in the normal activity of Wingless and
Hedgehog.

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Fig. 2. Glypicans are required for Wingless activity in embryos. (A-D) Expression
of rhomboid in various mutant embryos at about stage 13. (A)
Expression of rhomboid in wingless mutants is characterised
by a `tramtrack pattern' (this is seen in all embryos, n=100). (B) No
such pattern is seen in hedgehog mutants, which express
rhomboid in broadened stripes compared with wild type (stripes become
occasionally split as shown on the right hand side). (C) In wingless
hedgehog mutants, rhomboid stripes collapse into ventral rings
(this is true for most segments in all embryos, n=53). Such rings are
never seen in wingless mutants, n=100, and rarely so in
hedgehog mutants (12% of hedgehog mutants have one or two ventral
rings, none have more n=25). (D) The phenotype of embryos lacking
maternal and zygotic dally and dlp is similar to - though
slightly more variable than - that of wingless hedgehog mutants (a
majority of abdominal rhomboid stripes collapse into rings in 69% of
embryos and all embryos have at least one collapsed stripe, n=26).
(E) Cuticle phenotype of wingless hedgehog double mutant. The
epidermis is covered with denticles (the lawn phenotype) and two mid-ventral
`whorls' can be seen (arrows). Whorls are sometimes seen in hedgehog
mutants but not in the mid-ventral region. (F) The phenotype of embryos
lacking maternal and zygotic dally and dlp is similar
although more variable with fewer less marked mid-ventral whorls. This
suggests that weak residual signalling could occur in the absence of the
glypicans.
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Because dally dlp double mutant embryos are deficient in Hedgehog
signal transduction, they express only a small amount of wingless
during a short period [wingless transcription is maintained by
Hedgehog signalling (Lee et al.,
1992
)]. We therefore assayed the effect of artificially sustained
Wingless expression in the absence of Dally and Dlp. As shown in
Fig. 3, expression of
UAS-GFP-Wingless under the control of sim-gal4 leads to
local activation of Wingless signalling in dally dlp double mutants,
as evidenced by the formation of naked cuticle in the mid ventral region.
Therefore, dally and dlp are not absolutely essential for
Wingless signal transduction. Their roles in the embryo could be to boost or
sustain the signal, perhaps by ensuring sufficient retention/accumulation of
Wingless at cellular interfaces. In any case, the role of Dlp in Wingless
function seems to predominate because embryonic Wingless signalling appears
normal in the absence of Dally. However, Dally must provide some activity that
contributes to normal Wingless function because the phenotype of dally
dlp mutants is stronger than that of dlp single mutants.

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Fig. 3. Signal transduction in dally dlp mutants expressing exogenous
Wingless. (A) Lawn phenotype of an embryo lacking maternal and zygotic
dally and dlp, as shown in
Fig. 2E. (B) Naked cuticle is
induced along the ventral region by expression of GFP-Wingless under the
control of sim-gal4 in dally dlp-deficient embryos [as used by
Desbordes and Sanson (Desbordes and
Sanson, 2003 )]. This shows that signal transduction can take place
in the absence of the glypicans if sufficient Wingless expression is
sustained.
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At the prospective wing margin, dlp mutants display excess Wingless signalling while dally mutants suffer from a loss of Wingless signalling
To further investigate the roles of Dally and Dlp in Wingless function, we
analysed the phenotypes of adult mutants. As mentioned above, embryos lacking
all dally function are normal. In fact, they give rise to viable
adults. These have recognisable phenotypes in a variety of tissues, as
reported previously for presumed weaker alleles. This includes reduced and
rough eyes, disrupted antennae, loss of bristles and, in most males
(occasionally in females), loss of external genital structures
(Nakato et al., 1995
). We
concentrate on the wing because it allows a relatively simple assessment of
Wingless function. In wing imaginal discs, dally transcription is
upregulated near the Wingless source
(Nakato et al., 1995
),
suggesting a role at the prospective margin. As shown in
Fig. 4B,
dallyMH32 homozygotes display notches in the wing margin
at a low frequency (5%) (see also Nakato
et al., 1995
). Interestingly, the frequency and severity of these
notches is significantly higher (41%) in adults arising from maternal null
embryos (in an otherwise identical genetic background), suggesting that the
maternal contribution of dally is long lasting. Because loss of
margin tissue is indicative of insufficient Wingless signalling
(Phillips and Whittle, 1993
),
we conclude that wild type Dally contributes positively to Wingless
function.

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Fig. 4. Wing phenotypes in mutant flies. (A) Wild type. Homozygous dally
mutant flies survive and sometimes (5%) display notches in the margin, which
are symptomatic of reduced Wingless signalling (B). Distal truncation of vein
5 is also frequent but we have not attempted to characterise this further. The
few homozygous dlp mutants that survive to adulthood (around 1%) have
wings characterised by two fully penetrant phenotypes: a narrowing of the
space between veins 3 and 4 (C), which suggests reduced hedgehog
signalling (Crozatier et al.,
2002 ) and (D) the formation of ectopic bristles on either side of
the margin (arrowheads, compare with wild type shown in E), an indication of
excess Wingless signalling. The same phenotypes are seen in surviving flies
carrying the mutation (dlpMH20 and
dallyMH32) over a large deficiency (Df(3L) ED4543
dlp and Df(3L) ED4413 dally, respectively).
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Contrary to a previous report (Han et
al., 2004b
), we find that dlp is also not zygotically
required for adult viability: dlpMH20 homozygous flies
(with wild-type maternal contribution) survive to adulthood, albeit at a low
frequency. Therefore, the maternal contribution of dlp can support
development to adulthood, thus enabling us to assess the phenotype in wings.
Here, in situ hybridisation shows that dlp is uniformly expressed
except at the Wingless source where is it downregulated (not shown).
Surprisingly, dlpMH20 wings display a weak but fully
penetrant phenotype that is characteristic of apparent excess Wingless
signalling. During normal wing development, specialised bristles form at the
prospective wing margin, where Wingless signalling is highest during imaginal
disc development (Couso et al.,
1994
). In the wings of surviving dlpMH20
homozygotes, supernumerary bristles form on either side of the margin
(Fig. 4D), an indication of
ectopic Wingless signalling (Couso et al.,
1994
). Therefore, loss of dlp activity leads to excess
Wingless signalling, at least around the wing margin. It is intriguing that
mutations in dally and dlp lead to qualitatively different
phenotypes with respect to Wingless signalling at the wing margin, while these
two genes appear to be redundant for viability (homozygous double mutants die
at the end of embryogenesis, even in the presence of maternal contribution).
One possible explanation is that the two glypicans could perform distinct cell
biological functions that together contribute to optimal Wingless activity
(see below).
Overexpression of Dlp activates Wingless signalling in a non-cell-autonomous manner
In order to further investigate the specific roles of Dally and Dlp in
Wingless activity, we turned to overexpression experiments. When Dally is
overexpressed at 25°C with the dpp-gal4 driver, which is active
in a broad group of cells along the anteroposterior boundary in the wing, no
apparent effect on wing margin morphology can be seen (see also
Strigini and Cohen, 2000
). By
contrast, overexpressed Dlp induces clear-cut phenotypes. Overexpression of
Dlp in broad domains with engrailed-gal4 or dpp-gal4 at
25°C mainly causes loss of margin tissue, i.e. a reduction of Wingless
signalling there (Fig. 5A,B)
(see also Baeg et al., 2001
).
Likewise, scattered clones of misexpressing cells induced with the `Flp-on'
system (Pignoni and Zipursky,
1997
) cause margin loss (not shown). Oddly, however, an opposite
phenotype is also seen. As shown in Fig.
5C, the presence of random clones of cells that overexpress Dlp
causes the formation of ectopic margin bristles. Under all these experimental
conditions, Dlp overexpression induces a strong accumulation of Wingless at
the cell surface (only shown here for overexpression in clones, in
Fig. 5E,F) (see also
Baeg et al., 2001
). The
induction of opposite phenotypes (loss and gain of Wingless signalling) by
overexpression could be explained if Dlp had distinct cell-autonomous and
non-cell-autonomous effects. To investigate this further, mitotic clones of
Dlp-misexpressing cells were marked with the pwn mutation, which
affects the morphology of hairs and bristles
(Lawrence et al., 2002
). As
shown in Fig. 5D, ectopic
margin bristles are formed by wild-type cells located next to misexpressing
cells. This suggests that overexpressed Dlp causes non-cell-autonomous
increase in Wingless signalling. At the same time, it appears that Dlp
overexpression causes cell-autonomous reduction of Wingless signalling as
overexpression in a broad band of cells leads to loss of margin tissue.

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Fig. 5. Cell-autonomous and non-cell-autonomous effects of Dlp overexpression.
(A,B) Overexpression of Dlp in broad domains causes the loss of margin tissue.
Here, overexpression was activated in the posterior compartment with
engrailed-gal4 (A) or in the central region of the wing with
dpp-gal4 (B). Notching is localised to where the margin overlaps with
the region of overexpression (arrowheads). Details of the margin are shown on
the right-hand side. (C,D) Overexpression of Dlp in scattered clones (Flp-on
Dlp) leads to the formation of ectopic bristles (also to loss of margin
tissue, not shown here). (C) Ectopic bristles (arrows) caused by random
unmarked clones. (D) Margin area near a clone marked with the pwn
mutation (outlined). An ectopic bristle (pwn+, i.e.
outside the clone) is seen at the edge of the clone (arrow). (E,F)
Overexpression of Dlp causes local accumulation of Wingless at the cell
surface. Overexpression was induced in clones marked by ß-galactosidase
(E). Wingless accumulation is seen in F (visualised by anti-Wingless antibody)
(white arrows).
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Dlp overexpression reduces Wingless signalling near the margin while extending the range of low level signalling
The results described above lead to a model whereby, at the surface of a
given cell, Dlp would divert oncoming Wingless away from local signalling
while at the same time favouring export or presentation to neighbouring cells
(Fig. 6). According to such a
model, overexpressed Dlp would be expected to extend the range towards more
distant cells while at the same time reducing signalling near the source. This
prediction is indeed borne out. Within cells that overexpress Dlp,
senseless, a gene that responds to high level of Wingless
(Nolo et al., 2000
), is
repressed as expected from the loss of margin tissue. For example, in discs
overexpressing Dlp under the control of engrailed-gal4, senseless
expression is abolished in the posterior compartment (right side of the broken
line in Fig. 7A). At the same
time, the domain of expression of distal-less, a low level target
(Zecca et al., 1996
) is
broadened (Fig. 7B), suggesting
an extension of the range. A similar result is seen in large overexpression
clones (Fig. 7C,D). In this
figure, the slight upregulation of distal-less in cells flanking the
misexpressing clone (arrowhead), consistent with the possibility that
overexpressed Dlp favours presentation of Wingless to neighbouring cells.

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Fig. 6. Model that reconciles the opposite phenotypes seen as a result of
dlp overexpression. Wingless can bind to several receptors as it
reaches a target cell. Binding to Dlp would prevent access to the signalling
receptors and favour presentation to a neighbouring cell. By contrast, binding
to the signalling receptors would not only lead to signalling but also to
trapping and degradation, thus preventing subsequent transport.
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Fig. 7. Dlp overexpression blunts the Wingless gradient while loss of dlp
sharpens it. (A) Overexpression of Dlp with engrailed-gal4 eliminates
expression of senseless (a `high Wingless target') in the posterior
compartment (on the right of the broken line where engrailed-gal4 is
expressed). (B) At the same time, expression of distal-less, a `low
Wingless target' is broadened (arrowhead) specifically in the posterior
compartment. (C,D) A similar broadening of distal-less expression is
seen in large Dlp misexpression clones (right-hand clone, marked with GFP in
C). There is slight upregulation of distal-less at the edge of the
clone within its normal domain of expression (arrowhead in D) consistent with
increased presentation activity as a result of Dlp overexpression. (E,F)
Mutant dlp cells have reduced levels of Wingless protein. Mutant clones are
marked by the absence of GFP (green in E). Reduction of Wingless protein
(shown in F) is subtle but unambiguous [see, for example, the reduction in the
number of vesicles in the mutant area (arrowhead)]. (G,H) Reduction of
Wingless protein at the surface of dlp mutant cells. Extracellular
staining (shown in H) was performed as described previously
(Strigini and Cohen, 2000 ).
Again, mutant cells are marked by the absence of GFP (green in G). (I,J)
Expression of distal-less in wild type (I) and homozygous dlp (J)
discs. Both panels are from discs processed and photographed under identical
conditions. The domain of distal-less expression is clearly narrower
in wing discs obtained from dlp homozygous larvae than in the wild
type. Thus, in dlp mutants, a low level target is activated over a
reduced range. (K,L) Expression of senseless in wild-type (K) and
homozygous dlp (L) discs. Again, both panels are from discs processed
and photographed under identical conditions. The domain of senseless
expression is slightly wider in the dlp mutant, consistent with the
formation of ectopic bristles near the adult wing margin.
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Loss of dlp activity reduces the range of Wingless
If wild-type Dlp is indeed involved in shunting Wingless to neighbouring
cells, one expects that loss of dlp activity would reduce the range
of Wingless. This is indeed the case. Clones of dlp mutant cells
contain reduced amount of Wingless in receiving tissue (arrowhead in
Fig. 7F) without an apparent
reduction at the source. A reduction of Wingless present at the surface of
dlp mutant cells is also seen
(Fig. 7G,H). To assess the
functional consequence of reduced Wingless, we looked at the expression of
distal-less in imaginal discs obtained from homozygous
dlpMH20 larvae. The domain of distal-less
expression in dlp mutants is significantly narrower than that in
wild-type discs, suggesting a reduction of the range
(Fig. 7I,J). A concomitant
increase in senseless expression is seen
(Fig. 7K,L) suggesting
increased signalling near the source (as expected from ectopic margin
bristles, Fig. 4D). Thus, in
dlp mutants, the balance between short-range and long-range
signalling is upset, with short-range signalling being favoured at the expense
of long-range signalling.
Both Dally and Dlp bind Wingless in cell culture even though only Dlp overexpression causes Wingless accumulation in imaginal discs
Because dally mutants have reduced Wingless signalling, Dally
could act as a classical, though non-essential, co-receptor
(Ruoslahti and Yamaguchi,
1991
), perhaps capturing the ligand and presenting it to the
signalling receptors. To determine whether Dally does indeed promote binding
of Wingless to the cell surface, we used a tissue culture assay. S2 cells were
transfected with a plasmid encoding Dally. After a suitable time to allow
expression, cells were transferred to 4°C to prevent endocytosis and
treated with conditioned medium containing GFP-Wingless. As shown in
Fig. 8A,B, Dally expression
causes a significant increase in the accumulation of GFP-Wingless at the
surface of S2 cells. Transfection of dlp also causes Wingless
accumulation at the cell surface, possibly to a lesser extent
(Fig. 8C,D). We suggest that in
imaginal discs, after recruitment, Dally could present Wingless to signalling
receptors expressed in the same cell. This would be followed by activation of
signalling and rapid degradation. In imaginal discs, although exogenous Dlp
accumulates almost exclusively at the cell surface, Dally is present both at
the cell surface and in vesicles, perhaps because it is continuously
endocytosed (insets in Fig.
8E).

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Fig. 8. Binding activity and subcellular localisation of Dally and Dlp. (A-D)
Transfection of Dally-HA or Dlp-HA in S2 cells causes accumulation of
exogenous GFP-Wingless at the cell surface. Transfected cells are recognised
with anti-HA (in A and C). HA immunoreactivity is reproducibly lower for Dally
than for Dlp (compare A with C) but we do not know whether this is due to
differences in expression levels or epitope accessibility. Nevertheless, Dally
transfected cells reproducibly accumulate more GFP-Wingless (compare B with
D). (E,F) Subcellular distribution of exogenous Dally and Dlp in wing imaginal
discs. FLAG-tagged Dally expressed under the control of dpp-gal4 is
present both at the cell surface and in vesicles (E), while HA-tagged Dlp is
almost exclusively seen at the cell surface. Inset in E shows the detail of a
disc expressing FLAG-Dally that was briefly stained with Texas Red Dextran to
label the endocytic pathway. The disc was immersed live in a solution of Texas
Red dextran for 10 minutes. This was followed by a 20 minute chase and
subsequent fixation. Partial colocalisation of Dally (green) with dextran
(red) shows that some Dally is in endocytic structures.
|
|
 |
Conclusion
|
---|
The fact that mutations in dally and dlp cause different
phenotypes suggests that, although they both underpin Wingless function, these
two glypicans could perform distinct activities. It is likely that both Dally
and Dlp are able to capture Wingless at the surface of imaginal disc cells.
From the point of view of a given cell in vivo, Wingless captured by Dally
would be mostly destined for `internal consumption', while Dlp-bound Wingless
would be for export only. Subsequent long-range transport would occur by
hopping from Dlp on one cell to Dlp on the next. Both glypicans would
contribute to increasing the concentration of Wingless at the cell surface
(Dally in cis and Dlp in trans). We suggest that in the embryo too, Dlp and
Dally help in the presentation and reception of Wingless, respectively.
However, in this system, little Wingless transport takes place
(Pfeiffer et al., 2000
), maybe
because release of Wingless from Dlp is not allowed. It is interesting that,
in embryos, dlp is highly expressed in cells that secrete Wingless.
Therefore, the role of Dlp would mainly be to ensure that plenty of Wingless
is retained at the surface of Wingless-expressing cells thus allowing
sustained short-range signalling. In both the embryonic and disc systems, the
genetic redundancy between dally and dlp could be viewed as
follows: reduction of capturing activity in dally mutants would be
compensated by the `presentation activity' of Dlp and vice versa. Further cell
biological work will be needed to fully explore the specific activities of
Dally and Dlp and also to discover how Wingless is transferred from one cell
to another during transport, perhaps with the help of enzymes such as
Notum/Wingful (Gerlitz and Basler,
2002
; Giraldez et al.,
2002
).
 |
ACKNOWLEDGMENTS
|
---|
Xavier Franch-Marro is the recipient of a Wellcome Trust travelling
fellowship, Oriane Marchand and Eugenia Piddini are funded by EMBO and Marie
Curie fellowships, Sara Ricardo was funded by a Gulbenkian predoctoral
fellowship, while the others are supported by the Medical Research Council. We
acknowledge the Developmental Studies Hybridoma Bank, the Bloomington Stock
Center and various colleagues (listed in the Materials and methods) for
strains and antibodies. Special thanks to Toshiro Aigaki for the P-element
stocks used to generate the mutants, to Steve Cohen for the generous provision
of unpublished plasmids and strains, to Jose Casal for suggesting the
pwn experiment (Fig.
4D) and providing the necessary materials, and to John Roote and
Drosdel for stocks. David Wilkinson provided comments on the manuscript.
 |
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