1 Division of Developmental Biology, Cincinnati Children's Hospital Medical
Center, Cincinnati, OH 45229, USA
2 The Graduate Program in Molecular and Developmental Biology, University of
Cincinnati College of Medicine, Cincinnati, OH 45229, USA
Author for correspondence (e-mail:
xinhua.lin{at}cchmc.org)
Accepted 9 December 2004
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SUMMARY |
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Key words: Wingless (Wg), morphogen, Wnt, Heparan sulfate proteoglycans, Dally, Dally-like (Dlp), Frizzled (Fz) receptor, signaling
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Introduction |
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Wg is the founding member of the Wnt family of secreted proteins and plays
essential roles in many developmental processes
(Nusse, 2003;
Wodarz and Nusse, 1998
). In
the third instar wing disc, Wg is expressed in two to three cell widths
straddling the dorsoventral (DV) compartment boundary. Wg forms a
concentration gradient to activate the expression of its target genes
including achaete-scute (ac), distalless
(dll) and vestigial (vg) in a
concentration-dependent manner (Neumann
and Cohen, 1997
; Strigini and
Cohen, 2000
; Zecca et al.,
1996
). Studies from Drosophila embryonic epidermis and
the wing disc lead to several models to explain Wg movement across a field of
cells (Cadigan, 2002
;
Tabata and Takei, 2004
;
Teleman et al., 2001
;
Vincent and Dubois, 2002
). Two
prevalent models are: (1) diffusion through the extracellular space
(Strigini and Cohen, 2000
);
and (2) planar transcytosis via dynamin-mediated endocytosis
(Bejsovec and Wieschaus, 1995
;
Moline et al., 1999
). The
diffusion model proposes that Wg movement is through an extracellular route,
while planar transcytosis suggests that Wg moves via intracellular routes
through dynamin-mediated endocytosis. In addition, Wg movement by membrane
fragments called argosomes (Greco et al.,
2001
) or by retention on the cell surface
(Pfeiffer et al., 2000
;
Pfeiffer et al., 2002
) are
also proposed. In the wing disc, much of the evidence is consistent with the
diffusion model (Strigini and Cohen,
2000
). However, currently the exact mechanism(s) of Wg diffusion
and the molecules regulating this process are unclear.
Wg relays its signal through two functionally redundant receptors, Frizzled
(Fz) and Fz2, both of which belong to the family of seven-pass transmembrane
proteins (Nusse, 2003;
Wodarz and Nusse, 1998
). Arrow
(Arr), a member of the LDL receptor-related protein (LRP) family of proteins,
is also required for Wg signaling and has been postulated to act as a
co-receptor for Wg (He et al.,
2004
; Tolwinski et al.,
2003
; Wehrli et al.,
2000
). Previously, Cadigan et al. have proposed that Fz2 played
essential roles in shaping the Wg morphogen gradient in the wing disc
(Cadigan et al., 1998
). This
conclusion is mainly based on the following observations. First,
overexpression of Fz2 in the wing disc stabilizes Wg and expands the range of
Wg-target gene expression. Second, Fz2 expression in the wing disc is
inhibited by Wg signaling and appears to be an inverse gradient to that of Wg.
This inverse gradient of Fz2 is thought to facilitate the spread of Wg into
more distant areas. However, subsequent loss-of-function studies demonstrated
the functional redundancy of Fz2 and Fz for Wg signaling in both embryos and
in the wing disc (Bhanot et al.,
1999
; Chen and Struhl,
1999
). Therefore, the proposed role of Fz2 in Wg gradient
formation needs to be further examined by loss-of-function studies.
Furthermore, whether Fz and Arr are required for Wg gradient formation is
currently unclear.
In recent years, heparan sulfate proteoglycans (HSPGs) have been shown to
play roles in the distributions of morphogen molecules
(Esko and Selleck, 2002;
Lin, 2004
). HSPGs are the
cell-surface and extracellular matrix molecules composed of a protein core to
which heparan sulfate (HS) glycosaminoglycan (GAG) chains are attached
(Bernfield et al., 1999
;
Esko and Selleck, 2002
).
Glypicans represent the main cell-surface HSPGs that are linked to the plasma
membrane by a glycosyl phosphatidylinositol (GPI) linker. Drosophila
has two glypican members, Division abnormally delayed (Dally) and Dally-like
protein (Dlp) (Baeg et al.,
2001
; Khare and Baumgartner,
2000
; Nakato et al.,
1995
). Our recent studies have demonstrated that both Dally and
Dlp are required for Hh and Dpp movement through a restricted diffusion
mechanism (Belenkaya et al.,
2004
; Han et al.,
2004b
). Regarding Wg morphogen formation, several studies have
shown that mutations in genes involved in HS biosynthesis lead to defects in
Wg distribution in the wing disc (Baeg et
al., 2001
; Bornemann et al.,
2004
; Han et al.,
2004a
; Luders et al.,
2003
; Takei et al.,
2004
). We have previously shown that Dally plays a role in Wg
signaling in the wing disc (Lin and
Perrimon, 1999
). Furthermore, overexpression of Dlp leads to
enhanced extracellular Wg levels in the wing disc
(Baeg et al., 2001
). Together,
these studies implicate possible roles of Dally and Dlp in Wg gradient
formation; however, their functions and mechanisms in Wg gradient formation
have not been examined by loss-of-function study.
Additional evidence for the involvement of HSPGs in Wg distribution comes
from studies of notum (also called wingful)
(Gerlitz and Basler, 2002;
Giraldez et al., 2002
).
notum encodes a secreted protein that belongs to the
/ß-hydrolase superfamily with similarity to pectin
acetylesterases. In both embryos and wing discs, notum expression
mirrors wg expression. Mutations in notum lead to enhanced
Wg levels and increased Wg signaling, while overexpression of notum
blocks Wg signaling activity (Gerlitz and
Basler, 2002
; Giraldez et al.,
2002
). In Drosophila S2 cells, co-expression of Dally and
Dlp with Notum reduces the amount of Dally and altered the electrophoretic
mobility of Dlp, respectively. These data suggest that Notum may regulate Wg
signaling by modulating Dlp or/and Dally
(Giraldez et al., 2002
).
However, it remains to be determined whether Dally or Dlp or other HSPGs are
indeed the substrate(s) for Notum in vivo.
In this study, we attempt to systematically analyze the relative roles of the glypicans (Dally and Dlp), the Wg receptors (Fz and Fz2) and the Wg co-receptor Arr in Wg gradient formation in the wing disc. We show that Dally and Dlp are essential and have different roles in setting up the Wg gradient. The specificities of Dally and Dlp in Wg gradient formation are at least partially achieved by their distinct expression patterns. To our surprise, extracellular Wg levels are not reduced, but rather enhanced, in fz-fz2 or arr mutant cells. We further demonstrate that Wg protein fails to move across a stripe of dally-dlp mutant cells. Together, these new findings suggest that the Wg morphogen gradient in the wing disc is mainly controlled by Dally and Dlp. We propose that Wg establishes its concentration gradient by a restricted diffusion mechanism involving Dally and Dlp.
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Materials and methods |
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Antibodies and immunofluorescence
Fixation of imaginal discs and antibody staining procedures were performed
as described (Belenkaya et al.,
2002). Extracellular Wg staining was performed as described
(Baeg et al., 2001
;
Strigini and Cohen, 2000
).
Primary antibodies were used at the following dilutions: mouse anti-Wg 4D4 at
1:3 (Iowa Developmental Studies Hybridoma Bank; IDSHB), rat anti-Ci (1:10)
(Motzny and Holmgren, 1995
),
rabbit anti-ß-gal at 1:500 (Cappel), mouse anti-ß-gal at 1:3000
(Roche Molecular Biochemicals), mouse anti-Dlp at 1:50
(Lum et al., 2003
), rabbit
anti-Wg at 1:500 (Reichsman et al.,
1996
), mouse anti-Hnt at 1:50 (IDSHB), guinea pig anti-Hrs
(full-length) at 1:200 (Lloyd et al.,
2002
) and rabbit anti-GFP Alexa Fluor 488 at 1:1000 (Molecular
Probes). The primary antibodies were detected by fluorescent-conjugated
secondary antibodies from Jackson ImmunoResearch Laboratories.
Generation of marked clones and ectopic expression
Clones of mutant cells in the wing disc were generated by the FLP-FRT
method (Golic, 1991;
Xu and Rubin, 1993
) and
induced by subjecting first- or second-instar larvae to a heat-shock at
37°C for 1 hour. Ectopic expression of Dlp and Fz2-GPI in the P
compartment of the wing disc were induced by heat-shock at 30°C for 24
hours prior to dissection (see genotypes below). Mutations in Minute
on chromosomes 2R and 3L were used to generate large clones of cells mutant
for certain genes. The use of Minute-/+ does not perturb
normal Wg distribution (see Fig. S1 in the supplementary material). Below, we
list the genotypes used in our analyses.
Clones mutant for dally, dlp, dally-dlp and sflmarked by the absence of GFP (Figs 1, 8)
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Clones mutant for dsh, fz, fz2, fz1-fz2, sfl-fz1-fz2, arr, and arr-botv marked by the absence of GFP (Figs 2, 4, 5)
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Results |
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Extracellular Wg is not completely lost in either dally or
dlp mutant clones. We suspected that this may be due to the partially
redundant functions of Dally and Dlp, which was observed for Hh and Dpp
signaling (Belenkaya et al.,
2004; Han et al.,
2004b
). To test this, we examined the extracellular Wg levels in
clones mutant for both dally and dlp (referred to as
dally-dlp hereafter). Indeed, we observed a stronger reduction of the
extracellular Wg in the dally-dlp clones when compared with
dally or dlp clones (Fig.
1F-G'). This effect is independent of the positions of
dally-dlp clones. Collectively, these data suggest that Dally and Dlp
have both distinct and overlapping roles in shaping the Wg gradient.
Dlp forms an inverse gradient to the Wg gradient and its expression is down-regulated by Wg signaling
The distinct extracellular Wg defects associated with dally and
dlp clones suggest that Dally and Dlp may play different roles in Wg
gradient formation. One possibility is that Dally and Dlp may be
differentially expressed in the wing disc, and their levels may determine
their relative contributions to Wg gradient formation. We tested this
possibility by examining the expression patterns of Dally and Dlp in the wing
disc. Previously, Fujise et al. demonstrated that dally mRNA
distribution was virtually identical to that observed with a
dally-lacZ line (Fujise et al.,
2003). dally expression in the wing pouch is induced by
Hh and Wg signaling, but repressed by Dpp signaling
(Fujise et al., 2001
;
Fujise et al., 2003
). As shown
in Fig. 2A-A',
dally expression visualized by ß-galactosidase (ß-gal)
staining in the dally-lacZ line is higher in two regions in the wing
pouch. The first is a zone at the DV boundary, the width of which increases
with distance from the anteroposterior (AP) compartment boundary. The second
is a stripe of cells at the AP boundary, which corresponds to Hh signaling
cells (determined by Ci accumulation) (Fig.
2A').
We then examined Dlp expression using the anti-Dlp antibody
(Lum et al., 2003). In the
wing pouch, Dlp protein is distributed in most cells except in a zone centered
at the DV boundary (Fig.
2B,B'). This zone is about 7-10 cell diameters in width,
corresponding well to the region where removal of dlp does not alter
extracellular Wg levels (Fig.
1D-E'). As Dlp expression appears to be a gradient inverse
to the Wg gradient along the DV axis, we suspect that Dlp expression may be
negatively regulated by Wg signaling. Indeed, two lines of evidence support
this view. When Wg signaling is activated in random clones by overexpressing a
constitutively active form of Armadillo (Armact)
(Pai et al., 1997
), Dlp levels
are autonomously reduced within the clones
(Fig. 2C,C'). Conversely,
when Wg signaling is inhibited in random clones by overexpressing a
dominant-negative form of TCF (TCFDN)
(van de Wetering et al., 1997
)
or in clones mutant for disheveled (dsh)
(Nusse, 2003
;
Wodarz and Nusse, 1998
). Dlp
is induced autonomously within the clones
(Fig. 2D-E').
Collectively, our data argue that Dlp expression is negatively regulated by Wg
signaling. Taken together with the observation that Dally is upregulated by Wg
signaling at DV boundary (Fujise et al.,
2001
), our new results suggest that the relative contributions of
Dally and Dlp in Wg gradient formation are controlled, at least in part, by
their expression levels.
Notum modulates Wg signaling mainly by downregulating Dally activity in the wing disc
In the wing disc, Notum is expressed at the DV boundary and its expression
is induced by high levels of Wg signaling
(Gerlitz and Basler, 2002;
Giraldez et al., 2002
). Wg
signaling activity is enhanced in the notum mutant wing disc,
suggesting that Notum acts as an inhibitor for Wg
(Gerlitz and Basler, 2002
;
Giraldez et al., 2002
).
Although biochemical experiments demonstrated the ability of Notum in
modifying both Dally and Dlp in cultured S2 cells
(Giraldez et al., 2002
), it is
unclear whether Notum acts on Dally or Dlp in vivo to regulate Wg signaling in
the wing disc. As Dlp is absent or expressed at very low levels at the DV
boundary where Notum is expected to have the highest activity, we suspect that
high levels of Notum at DV boundary may downregulate Dlp protein levels,
thereby inhibiting Wg signaling. To test this possibility, we examined Dlp
protein levels in notum clones. The Dlp protein level is not enhanced
in a big clone mutant for notum
(Fig. 2E,E') and is also
not elevated in the notum homozygous wing disc (data not shown), demonstrating
that downregulation of Dlp protein levels at DV boundary is not due to Notum
activity.
As Dally is highly expressed at the DV boundary, we anticipated that Dally
may be a more relevant target for Notum in the wing disc. The following lines
of evidence support this view. First, we generated a double mutant for
dally and notum (dally-notum). If Dally is
downstream to, and the major substrate for, Notum, dally-notum mutant
phenotypes should resemble those of the dally mutant alone. Loss of
notum in the wing disc leads to upregulation of Wg signaling and
therefore induction of ectopic sense organ precursor (SOP) cells, which can be
visualized by Hindsight (Hnt) staining
(Giraldez et al., 2002)
(Fig. 3B), whereas loss of
dally leads to reduced numbers of SOP cells
(Fig. 3C). In
dally-notum mutant clones, we never saw ectopic Hnt staining.
Instead, we consistently observed reduced numbers of SOP cells in large clones
(Fig. 3D). Second, both
notum and dally homozygous animals can develop into adult
flies at a low frequency. As a result of elevated Wg signaling, the
notum wing bears many ectopic thick mechanosensory bristles and
curved chemosensory bristles at the wing margin
(Fig. 3F), whereas the
dally wings show a reduced numbers of chemosensory bristles
(Fig. 3G). In the
dally-notum clones, we could not find extra bristles but
rather observed a loss of chemosensory bristles
(Fig. 3H). Together, the
dally-notum mutant exhibits similar phenotypes to those seen in the
dally mutant, suggesting that Notum regulates Wg signaling mainly by
modifying Dally activity in the wing disc.
We further asked what role Dlp may play in Wg signaling. Previous work has
shown that ectopic expression of Dlp in the wing disc inhibits Wg signaling,
suggesting that Dlp may negatively regulate Wg signaling in the wing disc
(Baeg et al., 2001). Our
loss-of-function analysis suggests that this is indeed the case. Ectopic SOP
cells can occasionally be seen in dlp mutant clones
(Fig. 3I). Consistent with
this, escapers of dlp homozygous flies
(dlpA187/A203) show extra mechanosensory bristles
(Fig. 3K). However,
dlp is a weaker inhibitor of Wg signaling than notum, as
removal of dlp results in fewer ectopic SOP cells or extra bristles
than removal of notum. Importantly, we found that removal of both
notum and dlp phenocopies notum loss of function
(Fig. 3J,L). Together with the
absence/low level expression of Dlp at DV boundary, we argue that Notum acts
mainly on Dally to downregulate Wg signaling in the wing disc. Our data also
suggest that Wg downregulates Dlp expression to ensure its high levels of
signaling activity at the DV boundary.
Extracellular Wg levels are not reduced, but enhanced in the absence of Wg receptors (Fz and Fz2) or the co-receptor Arr
Next, we examined the roles of Wg receptors Fz and Fz2, as well as
co-receptor Arr in Wg morphogen gradient formation. The extracellular Wg
gradient is not altered in fz mutant clones in the late-third instar
larvae wing discs (Fig.
4A,A'). However, we occasionally observed slightly reduced
extracellular Wg levels in fz mutant clones in mid-third instar
larval discs (data not shown). This reduction must be transient because it is
of low frequency (13%, n=30) and disappears in late-third instar
discs. However, we never observed reduced extracellular Wg levels in
fz2 mutant clones (Fig.
4B,B'), suggesting that Fz2 is not essential for
extracellular Wg gradient distribution. Our loss-of-function results do not
support the previous view for a role of Fz2 in Wg distribution, which was
mainly based on ectopic expression data
(Cadigan et al., 1998). One
possibility is that Fz and Fz2 may be functionally redundant in extracellular
Wg distribution. To test this, we examined extracellular Wg levels in clones
mutant for both fz and fz2 (fz-fz2). To our
surprise, extracellular Wg levels are not reduced, but enhanced within the
mutant clones (Fig.
4C,C'). Wg refines its own expression domain at the DV
boundary and loss of Wg signaling in cells adjacent to the Wg-expressing cells
leads to ectopic Wg expression (Rulifson
et al., 1996
). This is likely to be the case for the enhanced
extracellular Wg in fz-fz2 cells adjacent to the DV boundary (yellow
arrows in Fig. 4C). However,
the increased extracellular Wg far away from the DV boundary (indicated by
turquoise arrows in Fig. 4C) in
fz-fz2 mutant cells is unlikely to be due to ectopic Wg expression as
these cells are more than six cells away from the Wg stripe at the DV
boundary. We also examined the role of the Wg co-receptor Arr in Wg gradient
formation. Similar to fz-fz2 clones, arr clones result in
accumulated extracellular Wg (Fig.
4D,D').
The increased extracellular Wg levels in fz-fz2 or arr
clones may be caused by at least two possibilities. First, as Dlp is repressed
by Wg signaling, fz-fz2 or arr clones may upregulate the
levels of Dlp, thereby indirectly enhancing the extracellular Wg levels.
Alternatively, removal of Wg receptor Fz and Fz2 or co-receptor arr
may impair the ability of cells to internalize Wg. The following lines of
evidence support the first possibility. First, as expected, Dlp is upregulated
in fz-fz2 or arr clones
(Fig. 5A,A',C,C'). Second, we examined extracellular Wg levels in clones mutant for
fz-fz2 and deficient for HSPGs. As the dlp and fz
loci are too close to each other, we were unable to generate a
dlp-fz-fz2 triple mutant chromosome. Instead, we used
sulfateless (sfl) which encodes a heparan sulfate
N-deacetylase/N-sulfotransferase required for HS biosynthesis
(Baeg et al., 2001). Mutations
in sfl are expected to impair most, if not all, functions of HSPGs,
including Dlp (Baeg et al.,
2001
; Lin and Perrimon,
1999
). Indeed, extracellular Wg is reduced in the
sfl-fz-fz2 clones (Fig.
5B,B'). Similarly, extracellular Wg is reduced in clones
mutant for both arr and botv, which encodes a heparan
sulfate co-polymerase required for HSPG biosynthesis
(Fig. 5D,D')
(Han et al., 2004a
;
Takei et al., 2004
). These
results suggest that enhanced extracellular Wg levels in
fz-fz2 or arr clones are HSPG dependant. Finally,
we also examined extracellular Wg levels in clones mutant for dsh.
Dsh is an intracellular protein acting downstream of the Wg receptor,
therefore it will probably not interfere with Wg internalization. dsh
mutant clones disrupt Wg signaling, thereby causing Dlp upregulation
(Fig. 2E,E'). Importantly, we observed very striking accumulation of extracellular Wg in
clones mutant for dsh (Fig.
5E-F'). Extracellular Wg accumulation can be clearly seen in
dsh clones which are located far away from DV boundary
(Fig. 5E-F').
Collectively, our results provide compelling evidence that accumulated
extracellular Wg protein in Fz-fz2 or arr mutant clones are
mainly resulted from upregulated Dlp, which probably further stabilizes
extracellular Wg protein on the cell surface.
We also examined internalized Wg vesicles in fz-fz2 or
arr clones by co-staining Wg and the endosome marker Hrs
(Lloyd et al., 2002). We
observed significant amount of internalized Wg vesicles in
fz-fz2 and arr clones, suggesting that Wg
internalization still occur in the absence of Fz and Fz2 activities or Arr
activity (data not shown).
The capacities of HSPGs (Dally and Dlp), the Wg receptors (Fz and Fz2) and the co-receptor Arr in modulating the extracellular Wg gradient
Previous studies and our loss-of-function data suggest that different HSPGs
and Wg receptors may have differential intrinsic abilities in influencing Wg
distribution. To compare their differences, we ectopically expressed
individual HSPGs and Wg receptors in the P compartment of the wing disc by
using enGal4 or hhGal4 drivers, and
then compared the extracellular Wg distribution in the A and P compartments.
The signal intensity of extracellular Wg staining was averaged and plotted
from two comparable regions selected from the A and P compartments. In the
wild-type disc, the patterns of the extracellular Wg gradient are virtually
identical in the A and P compartments (Fig.
6A-A'') although the extracellular Wg level is marginally
higher in the P compartment.
First, we ectopically expressed Dlp and Dally in the P compartment.
Persistent induction of UAS-dlp by enGal4 leads
to a greatly reduced size in the P compartment of the wing disc (data not
shown), presumably caused by interference of Wg signaling by Dlp. To overcome
the deleterious effect of early induction of UAS-dlp, we used a
temperature-sensitive allele of Gal80 (Gal80ts) to keep Gal4
inactive until the late stage of larval development. Gal80ts
functions as a repressor of Gal4 at the permissive temperature (19°C) but
allows Gal4 to be active at the non-permissive temperature (30°C)
(McGuire et al., 2003). When
UAS-dlp is induced by hhGal4 for 24 hours at the
non-permissive temperature (30°C) in the presence of Gal80ts,
extracellular Wg is significantly increased on the surface of Dlp
overexpressing cells and the visible range of the Wg gradient extends to the
whole wing pouch along the AP axis (Fig.
6B,B'). The plot profile suggests that the Wg gradient
becomes less steep and broader in the P compartment
(Fig. 5B''). Importantly,
the extracellular Wg level on the Wg-expressing cells is not significantly
changed, suggesting that the high levels of Dlp at the DV boundary does not
impede Wg movement. Similar results were obtained in the otherwise smaller
posterior half of the disc when enGal4 is used to induce
the expression of UAS-dlp (data not shown). Consistent with previous
results by others (Strigini and Cohen,
2000
), overexpression of Dally by enGal4 did
not drastically alter extracellular Wg levels
(Fig. 6C,C'). However,
the plot profile shows that there is a mild increase in the Wg level in the
region close to the DV boundary in the P compartment
(Fig. 6C''), although this
increase does not change the range and the steep shape of the Wg gradient
significantly.
Second, we examined the ability of the Wg receptors (Fz and Fz2) in
influencing the Wg gradient. Previous studies have shown that overexpression
of Fz-GPI has no effect on the Wg gradient when examined by conventional
staining (Rulifson et al.,
2000). However, we found that the extracellular Wg levels are
enhanced, and the Wg gradient becomes broader and flatter in the P compartment
when overexpressing wild-type Fz (Fig.
7A-A''). The apparent difference between our observations and
the previous work (Rulifson et al.,
2000
) most probably reflects a difference in the sensitivity of
detection methods rather than in the affinity of two versions of Fz for Wg.
Overexpression of Fz2, however, leads to increased Wg levels in the central
zone of the P compartment (Fig.
7B,B'). The plot profile shows that the range of the Wg
gradient is almost the same in the presence of a high level of Fz2
(Fig. 7B''), indicating
that the primary effect of ectopic Fz2 is stabilizing Wg rather than enhancing
Wg movement. We also found that Dlp levels are not altered in cells
overexpressing Fz or Fz2 (data not shown), suggesting that the enhanced
extracellular Wg levels are the direct result of overexpressing Fz and Fz2,
both of which can, as suggested by biochemical studies, directly bind to Wg
(Wu and Nusse, 2002
).
|
Wg moves from cell to cell in a HSPG-dependent manner
Our recent studies demonstrated that Dally and Dlp are required for Hh and
Dpp movement through a restricted diffusion mechanism
(Belenkaya et al., 2004;
Han et al., 2004b
). We
therefore examined whether Dally and Dlp control Wg movement through a similar
mechanism. We generated narrow stripes of clones mutant for sfl or
dally-dlp and asked whether Wg can move across these
HSPG-deficient cells. Extracellular Wg levels are reduced within the
sfl clones as well as in wild-type cells behind the clones
(Fig. 8A-B', shown by
turquoise arrows), even though these clones are only one to two cell diameters
wide. Similar results were obtained for dally-dlp clones
(Fig. 8C-D'), suggesting
that Wg movement is impeded by cells mutant for both dally and
dlp. We also quantified this cell non-autonomous effect by generating
profiles of Wg distribution in a region covering both the clones and wild-type
areas behind the clones. The plots show that extracellular Wg is reduced to
basal levels in the wild-type cells behind the sfl or
dally-dly mutant clones (Fig.
8A'',B'',C'',D''). Collectively, these data
argue that Wg movement is not by free diffusion, but rather is mediated by a
restricted diffusion involving the HSPGs Dally and Dlp.
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Discussion |
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The glypicans Dally and Dlp in Wg gradient formation
One important finding in this work is that the glypicans Dally and Dlp are
required for Wg gradient formation. Several recent studies have shown that
extracellular Wg distribution is compromised in clones mutant for HS
biosynthesis enzymes, including sfl, slalom and members of the
Drosophila EXT gene (Baeg et al.,
2001; Bornemann et al.,
2004
; Han et al.,
2004a
; Luders et al.,
2003
; Takei et al.,
2004
). However, it is unclear which HSPG cores are involved in
this process. We show that Wg morphogen distribution is defective in either
dally or dlp mutant clones. These new findings clearly
establish the requirement of Dally and Dlp in Wg morphogen gradient formation.
Thus, as in the case of Hh and Dpp
(Belenkaya et al., 2004
;
Han et al., 2004b
), the
glypican members Dally and Dlp, rather than Drosophila syndecan or
perlecan, are the main HSPGs involved in Wg gradient formation.
Interestingly, we found that Dally and Dlp differentially regulate the Wg
extracellular gradient in distinct regions of the wing disc. Both Dally and
Dlp are glypican members of HSPG family. One would expect that differences in
the structure of Dally and Dlp, and their attached HS GAG chains may determine
their abilities to interact with Wg, thereby leading to their specificities.
This is probably one of the factors, as overexpression of Dally and Dlp has
very different effects on extracellular Wg gradient. Consistent with our data
in this work, previous studies have shown that Dlp is much more potent in
accumulating Wg protein than Dally when overexpressed
(Baeg et al., 2001;
Giraldez et al., 2002
).
However, we have also found that the regional effects of Dally and Dlp on
extracellular Wg gradient correspond well to their expression patterns. The
regions with higher expression levels of Dally or Dlp have stronger
extracellular Wg defects when Dally or Dlp is removed, respectively. Based on
these data, we suggest that the differential roles of Dally and Dlp in
extracellular Wg distribution are at least partially determined by their
restricted expression.
What exact roles do Dally and Dlp play in shaping the extracellular Wg gradient? Our loss-of-function results suggest that removal of Dally or Dlp leads to reduced extracellular Wg levels on the cell membrane. Furthermore, extracellular Wg levels are reduced in wild-type cells behind sfl or dally-dlp clones (Fig. 8). These data suggest that the primary function of Dally and Dlp in Wg gradient formation is to maintain extracellular Wg proteins so that locally concentrated Wg proteins can further move to more distal cells through diffusion.
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Roles of Dlp, Dally and Notum in modulating Wg signaling |
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Previous studies have identified Notum as a secreted inhibitor for Wg
signaling (Gerlitz and Basler,
2002; Giraldez et al.,
2002
). Notum is expressed at the DV boundary and was proposed to
downregulate Wg signaling by modulating Dlp activity
(Giraldez et al., 2002
).
During the reviewing process of this manuscript, Cohen's
(Kreuger et al., 2004
) and
Selleck's (Kirkpatrick et al.,
2004
) laboratories published their recent studies proposing that
Notum negatively regulates Wg signaling by shedding of Dlp, which converts Dlp
from a membrane-tethered co-receptor to a secreted antagonist. Their
conclusions are mainly based on two lines of experimental data. First,
biochemical experiments clearly demonstrated that Notum can modify Dlp in a
manner that resembles cleavage of the GPI anchor
(Kreuger et al., 2004
).
Second, Kirkpatrick et al. showed that transheterozygous dlp/notum
flies produced ectopic mechanosensory bristles which are not seen in
dlp+/- or notum+/- alone, indicating
that Dlp and Notum genetically collaborate in downregulating Wg signaling
(Kirkpatrick et al.,
2004
).
However, on the basis of our data in this work, we suggest that Notum
inhibit Wg signaling mainly by modifying Dally in the wing disc. First,
genetic interaction data shown by Kirkpatrick et al. cannot distinguish
whether Dlp and Notum work in the same pathway or in two independent pathways
to downregulate Wg signaling at the DV boundary
(Kirkpatrick et al., 2004). If
Dlp is indeed the main substrate for Notum, we would expect that ectopic Wg
signaling activity in dlp-notum should be similar to that in
dlp mutant. However, our loss-of-function analysis demonstrates that
ectopic Wg signaling in dlp-notum is similar to that in
notum mutant, but much stronger than that in dlp mutant.
However, dally-notum clones exhibits loss of Wg signaling activity,
which is similar to dally mutant. Second, Dlp expression is
strikingly repressed by Wg signaling and this reduction is independent of
Notum. Low/absent expression of Dlp is not consistent with the view that Dlp
is the main substrate for Notum. Finally, it is important to mention that
Notum can reduce the amount of Dally when they are co-expressed in
Drosophila S2 cells (Giraldez et
al., 2002
), suggesting that Notum can modify Dally as well.
Although Notum can shed Dlp, whether shed Dlp acts as a Wg inhibitor need to
be further determined (Kreuger et al.,
2004
). Therefore, further experiments are necessary to define the
mechanism(s) of Notum-mediated Wg inhibition.
Roles of Fz, Fz2 and Arr in Wg gradient formation
One important finding of this study is that removal of the Wg receptors (Fz
and Fz2) and the co-receptor Arr does not lead to a loss of extracellular Wg.
Fz2 has been proposed to play a major role in Wg gradient formation in the
wing disc by ectopic expression studies
(Cadigan et al., 1998).
Although several previous studies as well as our data in this work
demonstrated the high capacity of Fz2 in stabilizing Wg
(Cadigan, 2002
;
Lecourtois et al., 2001
;
Rulifson et al., 2000
), our
loss-of-function results clearly show that extracellular Wg levels were not
reduced in clones mutant fz2. This is apparently not due to the
overlapping function of Fz as the extracellular Wg level is enhanced, rather
than reduced in the absence of both Fz and Fz2 functions. Our results argue
that Fz2 is not essential for extracellular Wg gradient formation in vivo. It
is important to note that in addition to Fz and Fz2, Drosophila Fz3
is also expressed in the wing disc and its expression is upregulated by Wg
signaling (Sato et al., 1999
).
Although Fz3 has lower affinity than Fz2 in Wg binding and acts as an
attenuator of Wg signaling (Sato et al.,
1999
; Wu and Nusse,
2002
), its role in Wg distribution needs to be determined.
We further demonstrated that extracellular Wg is enhanced in cells mutant for fz-fz2 or arr, suggesting that Wg receptors (Fz and Fz2) and Arr shape extracellular Wg gradient by downregulating extracellular Wg levels. Our data argue that this is mainly resulted from upregulation of Dlp. Consistent with this view, we show that the accumulated extracellular Wg can be eliminated by loss of HSPGs in sfl-fz-fz2 or arr-botv mutant clones (Fig. 5). Importantly, we show that both extracellular Wg and Dlp levels are upregulated on the cell surface of clones mutant for dsh. These data provide compelling evidence that though a feedback mechanism, Wg signaling can control the Dlp levels to regulate the extracellular Wg gradient.
Another alternative possibility is that enhanced Wg levels in
fz-fz2 or arr clones may be caused by impaired Wg
internalization. Although we observed significant amount of internalized Wg
vesicles in fz-fz2 or arr mutant clones (data not shown), we
cannot rule out this possibility as a quantitative comparison of Wg
internalization between wild-type cells and fz-fz2 or arr
mutant cells was difficult in our experimental settings. Furthermore, as
mentioned above, Fz3 is expressed in the wing disc and its expression is
upregulated by Wg signaling (Sato et al.,
1999). It is possible that Fz3 may mediate the internalization of
Wg in the absence of Fz and Fz2.
Molecular mechanisms of Wg movement
A previous study by Strigini and Cohen provided evidence that Wg morphogen
movement is regulated by a diffusion mechanism(s) in the wing disc
(Strigini and Cohen, 2000).
Does Wg diffuse freely in the extracellular matrix/space? In this work, we
show that Wg fails to move across a strip of cells mutant for the HSPGs Dally
and Dlp. This result suggests that Wg can not freely diffuse in the
extracellular matrix. Instead, our findings are consistent with a model in
which Wg movement is mediated by the HSPGs Dally and Dlp through a restricted
diffusion along the cell surface (Fig.
9). Similar mechanisms have been proposed for Hh and Dpp
(Belenkaya et al., 2004
;
Han et al., 2004b
). In
biological systems such as imaginal discs, the restraint of Wg spreading to
the surface of the epithelial cell layer is important as the folding of
imaginal discs, such as the leg disc, poses a problem if the Wg gradient
formation were to occur out of the plane of the epithelial cell layer through
free diffusion. In agreement with this view, our model proposes that Wg
gradient formation depends on Wg movement through the cell surface of the disc
epithelium.
|
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ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/4/667/DC1
* These authors contributed equally to this work
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