Dally regulates Dpp morphogen gradient formation in the Drosophila wing
Momoko Fujise1,*,
,
Satomi Takeo1,*,
Keisuke Kamimura1,2,
Takashi Matsuo1,
Toshiro Aigaki1,
Susumu Izumi1 and
Hiroshi Nakato2,
1 Department of Biology, Tokyo Metropolitan University, Hachioji-shi, Tokyo
192-0397, Japan
2 Department of Molecular and Cellular Biology, and Arizona Cancer Center,
University of Arizona, Tucson, AZ 85724, USA
Present address: Institute for Molecular Science of Medicine, Aichi Medical
University, Nagakute, Aichi 480-1195, Japan
Author for correspondence (e-mail:
hnakato{at}azcc.arizona.edu)
Accepted 8 January 2003
 |
SUMMARY
|
---|
Decapentaplegic (Dpp), a Drosophila TGFß/bone morphogenetic
protein homolog, functions as a morphogen to specify cell fate along the
anteroposterior axis of the wing. Dpp is a heparin-binding protein and Dpp
signal transduction is potentiated by Dally, a cell-surface heparan sulfate
proteoglycan, during assembly of several adult tissues. However, the molecular
mechanism by which the Dpp morphogen gradient is established and maintained is
poorly understood. We show evidence that Dally regulates both cellular
responses to Dpp and the distribution of Dpp morphogen in tissues. In the
developing wing, dally expression in the wing disc is controlled by
the same molecular pathways that regulate expression of thickveins,
which encodes a Dpp type I receptor. Elevated levels of Dally increase the
sensitivity of cells to Dpp in a cell autonomous fashion. In addition,
dally affects the shape of the Dpp ligand gradient as well as its
activity gradient. We propose that Dally serves as a co-receptor for Dpp and
contributes to shaping the Dpp morphogen gradient.
Key words: dally, dpp, Morphogen gradient, Heparan sulfate proteoglycan, Drosophila
 |
INTRODUCTION
|
---|
Morphogens specify different cell fates in a concentration-dependent manner
and their functions are fundamental to tissue patterning during development.
In spite of the central role morphogens play in assembling tissues, the
molecular mechanisms of morphogen gradient formation remain largely unknown.
During development of the Drosophila wing, three molecules have been
proposed to act as morphogens: Wingless (Wg), Hedgehog (Hh) and
Decapentaplegic (Dpp), a homolog of vertebrate bone morphogenetic proteins
(BMPs). Dpp is expressed in a stripe of cells adjacent to the anterior
(A)-posterior (P) compartment boundary (A/P border cells), and patterns the
wing by inducing different target genes, such as spalt and
optomotor-blind, at different extracellular concentrations
(Lecuit et al., 1996
;
Nellen et al., 1996
).
Recently, two molecular tools have been developed to monitor the Dpp gradient
formation. First, a biologically active GFP-tagged Dpp (Dpp-GFP) was used to
directly visualize the Dpp gradient in the developing wing
(Fig. 1A)
(Entchev et al., 2000
;
Teleman and Cohen, 2000
).
Studies using Dpp-GFP successfully demonstrated that Dpp forms a long-range
gradient throughout the wing pouch. Dpp-GFP moves quickly through the disc and
is rapidly turned over. Second, an antibody that specifically recognizes the
phosphorylated form of Mothers against Dpp (pMad) can be used to monitor the
Dpp morphogen activity gradient by following the phosphorylation of Mad, a
downstream transducer of the Dpp pathway
(Fig. 1B)
(Persson et al., 1998
;
Tanimoto et al., 2000
).
Generally, pMad levels are high at the central region of the wing disc and
gradually decline toward the anterior and posterior distal cells. However,
within the central region, pMad levels are lower at the A/P border cells owing
to the reduced expression of thickveins (tkv), which encodes
the type I receptor for Dpp. Although the ligand and activity gradients now
can be visualized by using the tools mentioned above, the molecular basis for
the Dpp gradient formation is not yet completely understood.

View larger version (122K):
[in this window]
[in a new window]
|
Fig. 1. Dpp morphogen gradients and expression of Dpp signaling components in the
developing wing. (A,B) Patterns of Dpp-GFP expressed in A/P border cells by
dpp-GAL4 driver (A; dppd12/+;
dpp-GAL4/UAS-Dpp-GFP) and Dpp activity gradient visualized by anti-pMad
antibody staining (B). (C,D) Expression patterns of the Dpp type I receptor
gene tkv (C) and a putative Dpp co-receptor gene dally (D),
were followed by anti-ß-galactosidase antibody staining of wing discs of
the enhancer trap lines tkvP906 and
dallyP2, respectively. Brackets show positions of the A/P
border cells. Posterior is towards the right and ventral is towards the top of
all wing discs and in all subsequent figures.
|
|
One of the determinants of the Dpp morphogen gradient is the Tkv receptor.
As Dpp signaling negatively regulates tkv expression, the relative
levels of tkv are high in cells at the peripheral region of the wing
disc and are low within the central domain
(Fig. 1C) (Lecuit and Cohen, 1998
). In
addition, tkv expression is strongly repressed by Hh signaling at the
A/P border cells, which results in a reduction of pMad staining in this
region. The basal level of tkv expression is higher in the P
compartment than in the A compartment and is maintained by the activity of the
P cell-specific selector gene, engrailed (en)
(Funakoshi et al., 2001
;
Tanimoto et al., 2000
). As
higher levels of Tkv limit the movement of Dpp, Dpp does not spread as far in
the P compartment, resulting in a steeper Dpp morphogen gradient.
Overexpression of tkv was also shown to retard the movement of Dpp
(Lecuit and Cohen, 1998
;
Tanimoto et al., 2000
). Thus,
Dpp gradient formation is in part controlled by the regulated expression of
the tkv gene.
Several lines of evidence have suggested that heparan sulfate proteoglycans
(HSPGs) are involved in distribution of morphogens. tout velu
(ttv), a Drosophila homolog of the mammalian EXT tumor
suppressor gene family, encodes a heparan sulfate co-polymerase
(Bellaiche et al., 1998
;
The et al., 1999
;
Toyoda et al., 2000
).
Mutations in ttv disrupt the movement of Hh from its site of
production. Thus, ttv-dependent synthesis of HSPG is required for
normal distribution of Hh between cells. Similarly, Notum (Wingful) was
recently reported to be required for gradient formation of the Wg morphogen
(Gerlitz and Basler, 2002
;
Giraldez et al., 2002
). This
gene encodes a protein with homology to pectin acetyltransferases and is
proposed to affect Wg distribution by modulating the structures of the heparan
sulfate moiety of HSPGs. Recently, a novel membrane exovesicle structure,
argosome, was identified and proposed to be involved in morphogen movements
(Greco et al., 2001
). The
transportation of argosomes across cells and the localization of Wg protein in
this structure suggests a possible role for argosomes in Wg distribution.
Interestingly, when discs were treated with heparitinases, enzymes that digest
heparan sulfate, Wg failed to accumulate properly in argosomes, although
argosome distribution was not affected. Greco et al. proposed from this
observation that interaction of Wg with HSPGs is required for the
incorporation of Wg into argosomes (Greco
et al., 2001
). These findings collectively point to the
possibility that HSPGs are involved in controlling the distributions and
activities of morphogens during tissue patterning.
One of the Drosophila proteoglycan genes that has been shown to
affect growth factor signaling in tissue patterning is division abnormally
delayed (dally) (Nakato et
al., 1995
). dally encodes the core protein for glypican,
a family of HSPGs that are linked to the cell membrane through a GPI anchor
(Tsuda et al., 1999
).
dally affects signaling mediated by two known HS-binding growth
factors in Drosophila: Dpp and Wg. Modulation of dally gene
dosage dramatically alters the expression of Dpp target genes, as well as the
patterning activity of Dpp in multiple imaginal tissues
(Fujise et al., 2001
;
Jackson et al., 1997
). In the
embryonic epidermis, dally modulates patterning directed by Wg
(Lin and Perrimon, 1999
;
Tsuda et al., 1999
). These
findings are consistent with a model in which HSPGs enhance the activity of
growth factors on the cell surface by promoting the assembly and/or increasing
the stability of signaling complexes. In this study, we focused on the
function of dally in Dpp signaling during wing development. We found
that elevated levels of dally increase the sensitivity of cells to
Dpp, and that alterations in levels of dally affect formation of both
Dpp ligand and activity gradients. In addition, we found that the same
regulatory networks control expression of dally and tkv.
These findings suggest that the regulated expression and function of Dally are
essential components for Dpp morphogen gradient formation.
 |
MATERIALS AND METHODS
|
---|
Fly stocks
Enhancer trap lines, dallyP2
(Fujise et al., 2001
;
Nakato et al., 1995
) and
tkvP906 (Tanimoto et
al., 2000
) were used to follow the expression patterns of
dally and tkv, respectively. The mutations and transgenic
animals used were as follows: dallygem and
dallyDP-527, hypomorphic alleles of
dally (Nakato et al.,
1995
); Df enE, an embryonic lethal allele of
en (Tabata et al.,
1995
); tkva12
(Nellen et al., 1994
;
Nellen et al., 1996
) and
tkv6 (Singer et al.,
1997
), a null and hypomorphic allele of tkv,
respectively; UAS-dally (Tsuda et
al., 1999
); UAS-hhCD2, a membrane-tethered form of Hh,
the signaling fragment of Hh fused to the rat CD2 gene
(Strigini and Cohen, 1997
);
UAS-tkvQ253D, a constitutively active form of tkv
(Nellen et al., 1996
;
Wieser et al., 1995
);
Act5C>y+>GAL4, a FLP-OUT cassette to misexpress the
gene downstream of UAS (Ito et al.,
1997
); UAS-Dpp-GFP, a biologically active form of Dpp
tagged with GFP (Teleman and Cohen,
2000
); and dpp-GAL4
(Morimura et al., 1996
).
Immunostaining
Immunostaining was performed as previously described
(Fujise et al., 2001
) using
rabbit anti-ß-galactosidase (1:500, Cappel), rabbit anti-pMad [1:1000, a
generous gift from T. Tabata and P. ten Dijke
(Persson et al., 1998
;
Tanimoto et al., 2000
)] and
rat anti-Tkv [1:250, a generous gift from S. Cohen
(Teleman and Cohen, 2000
)].
The intensity profiles of pMad staining were generated by NIH Image using the
plot function. The primary antibodies were detected with Alexa Fluor 568- or
Alexa Fluor 488-conjugated secondary antibodies (Funakoshi).
Ectopic expression
Clones of cells that ectopically express hhCD2,
tkvQ253D and dally were induced by the GAL4/UAS
system using a FLP-OUT cassette (Ito et
al., 1997
; Struhl and Basler,
1993
). The genotypes of the flies used were:
- y w hsp70-flp/+; Act5C>y+>GAL4 UAS-GFP/+;
UAS-hhCD2/dallyP2,
- y w hsp70-flp/+; Act5C>y+>GAL4 UAS-GFP/+;
UAS-tkvQ253D/dallyP2 and
- y w hsp70-flp/+; Act5C>y+>GAL4 UAS-GFP/+;
UAS-dally/+.
Mosaic analyses
Homozygous mutant clones were induced by FLP-mediated mitotic recombination
(Golic, 1991
;
Xu and Rubin, 1993
). Larvae
were heat shocked at 24-48 hours after egg-laying at 37°C for 10-60
minutes to induce recombination. Discs were dissected and analyzed 48-96 hours
after the induction. The genotypes of larvae we used to examine the effect of
tkv mutations on dally::lacZ expression were: Ubi-nlsGFP
FRT40/tkva12 FRT40; dallyP2/FLP3 Sb
MKRS and Ubi-nlsGFP FRT40/tkv6 FRT40;
dallyP2/FLP3 Sb MKRS. To induce en
mutant clones, we used discs from y w hsp70-flp/+;
FRT42D y+ Df enE/FRT42D y+
Ubi-GFP; dallyP2/+ larvae.
Expression and detection of Dpp-GFP
To monitor the Dpp-GFP distribution, we dissected wing discs of
dppd12/+; UAS-dpp-GFP/dpp-GAL4 larvae. Distribution of
Dpp-GFP in dally mutants was observed using discs from
dppd12/+; dpp-GAL4 dallygem/UAS-dpp-GFP
dallygem larvae. The effect of dally overexpression
in A/P border cells on Dpp-GFP distribution was analyzed using discs from
dppd12/+; UAS-dpp-GFP UAS-dally/dpp-GAL4 larvae. In
previous studies (Entchev et al.,
2000
; Teleman and Cohen,
2000
), Dpp-GFP was expressed in dpp homozygous animals
(dppd8/dppd12 or
dppd12/dppd16). However, because animals
homozygous for both dpp and dally rarely survive to the
third instar, we expressed Dpp-GFP in a dpp heterozygous background
(dppd12/+). After a brief fixation (10 minutes) of discs
with 4% formaldehyde, signals for Dpp-GFP were imaged using confocal
microscopy (LSM410, Carl Zeiss). Average intensity profiles for different
genotypes were generated using NIH Image.
 |
RESULTS
|
---|
Expression pattern of dally along the AP axis of the wing
disc
The expression pattern of dally, monitored by dally::lacZ
enhancer-trap expression, in the developing wing along the AP axis shows a
peak of expression at the A/P border cells, and dally levels are
lowest in cells adjacent to this region
(Fig. 1D). Furthermore,
dally levels gradually increase toward the anterior and posterior
distal cells. This pattern correlates with the expression patterns of several
genes involved in pattern formation along the AP axis, such as tkv
and master of thick veins (mtv; Sbb
FlyBase), which suggests that dally also participates in this
process. Expression of the tkv gene is controlled by two distinct
pathways. First, Hh represses tkv expression at the A/P border cells,
and En regulates a high basal level of tkv in the P compartment
(Funakoshi et al., 2001
;
Tanimoto et al., 2000
). The
activities of both hh and en genes are mediated by a
putative transcription factor, Mtv. Second, tkv levels are
downregulated by Dpp signaling (Lecuit and
Cohen, 1998
). By this mechanism, tkv expression is
maintained at low levels in the center of the disc and at higher levels toward
the anterior and posterior edges. The correlation between expression patterns
of dally and tkv prompted us to analyze the dally
function in Dpp signaling in this tissue. As a first step toward this, we
analyzed the regulatory pathways controlling dally expression and
compared them with those controlling tkv expression.
dally expression is regulated by Hh and En
In a previous study, we showed that Hh signaling induces dally
expression at the A/P border cells (Fujise
et al., 2001
). dally expression was absent in the
smoothened (smo) mutant clones generated in the A
compartment, where the Hh signaling is blocked
(Alcedo et al., 1996
),
indicating that Hh signaling is required for activation of dally at
the A/P border cells. To further determine whether Hh signaling is sufficient
for the induction of dally, we examined clones that ectopically
express hhCD2, which encodes a membrane-tethered form of Hh
(Strigini and Cohen, 1997
),
using the FLP-OUT system. In the A compartment, dally expression
levels were increased in hhCD2-expressing cells and in cells
immediately adjacent to them (Fig.
2A-C). This result shows that Hh expression is sufficient to
induce dally expression in the A compartment. To determine if
dally expression is controlled by en, which upregulates
tkv expression, we induced clones of en-mutant cells using
the FLP-FRT mosaic analysis system. Within en-mutant clones in the P
compartment, dally levels were dramatically increased
(Fig. 2D-F), which indicates
that dally expression is negatively regulated by en.

View larger version (113K):
[in this window]
[in a new window]
|
Fig. 2. Expression of dally is regulated by Hh and En. (A-C) Misexpression
of the membrane-tethered form of Hh, HhCD2, induces ectopic expression of
dally. dally::lacZ expression was followed using
anti-ß-galactosidase antibody staining (A). Clones expressing
hhCD2 are marked by GFP in (B). (C) A merged image of A and B. Levels
of dally::lacZ are increased both in cells inside and
adjacent to the clones located in the A compartment (arrows), while clones in
the P compartment do not alter dally expression. (D-F) dally
is repressed by en in the P compartment. (D) Expression pattern of
dally::lacZ. (E) Positions of en-mutant clones are shown by
loss of GFP signal. (F) A merged image of D and E. dally expression
is significantly elevated in en-mutant clones induced in the P
compartment (arrow), indicating that dally is normally downregulated
by en. Arrowheads in A and D indicate the AP boundary.
|
|
Expression of dally enhancer trap is repressed by Dpp
signaling
To determine whether modulation of Dpp signaling affects dally
expression, we first compared the dally::lacZ expression between
wild-type and tkv heterozygous cells. Clones mutant for tkv
were generated in a heterozygous background (tkva12/+)
using the FLP-FRT system, which should, as a consequence, produce both mutant
(tkva12/tkva12) and wild-type sister clones
(+/+). However, tkv cells do not survive in the
wing pouch as Tkv activity is indispensable for growth and, thus, only
wild-type sister clones survived (Burke and
Basler, 1996
). In resultant mosaic discs with wild-type and
tkv-heterozygous cells, dally expression was decreased cell
autonomously in wild-type (+/+) clones at the AP border
(Fig. 3A-C) and peripheral to
the border (Fig. 3D-F). To
further confirm this result, we also examined the effect of
tkv-hypomorphic clones (tkv6/tkv6) on
dally expression. In such clones, where tkv activity is
partially compromised, the levels of dally expression were elevated
(Fig. 3G-I). In the notum
region of the wing disc, we could generate tkv-null clones
(tkva12/tkva12) in which a substantial increase
of dally expression was observed
(Fig. 3J-L). Finally, we tested
the effect of increased Dpp signaling on dally expression by using
the FLP-OUT method to induce clones of cells that express
tkvQ253D, a constitutively active form of tkv, in
the wing pouch. We found that the level of dally::lacZ expression was
autonomously reduced in the tkvQ253D-expressing clones
(Fig. 3M-O). All of these
results consistently indicate that dally expression in the wing disc
is negatively regulated by Dpp signaling, as has been shown for tkv
(Lecuit and Cohen, 1998
).
Thus, dally and tkv are regulated by the same set of
molecular pathways: Hh, En and Dpp signaling.

View larger version (91K):
[in this window]
[in a new window]
|
Fig. 3. Expression of dally is repressed by Dpp signaling. (A-F) Clonal
analysis for a severe tkv allele, tkva12,
resulted in loss of the clones homozygous for tkv, because of growth
defects, and preferential growth of their sister clones carrying two copies of
the wild-type tkv in a genetic background heterozygous for
tkv (one copy of wild-type tkv). The sister clones are
marked by two copies of GFP (+/+), giving brighter signals than the single
copy of GFP (+/) (B,E). Wild-type (+/+) clones at the A/P border (A-C)
and peripheral to the border (D-F) autonomously decrease the expression of
dally::lacZ (arrows). (G-I) Clonal analysis for a mild
tkv allele. dally::lacZ expression (G) is elevated in clones
of cells homozygous for a partial loss-of-function tkv allele,
tkv6 (arrows). (J-L) Effects of tkva12
mutation on dally::lacZ expression in the notum. Levels of
dally expression (J) were significantly increased in
tkva12 clones (arrows). Positions of clones are shown by
loss of the GFP signal (H,K). (M-O) Effects of ectopically activated Dpp
signaling on dally::lacZ expression. Expression of dally was
repressed in the FLP-OUT clones that express a constitutively active form of
tkv, tkvQ253D (M). Positions of the FLP-OUT clones are
marked by GFP (N). (C,F,I,L,O) Merged images of the two left-hand images on
each row.
|
|
dally affects the sensitivity of cells to Dpp
Although Dally was shown to be involved in Dpp signaling in several
imaginal tissues (Jackson et al.,
1997
), the function of Dally in this signaling pathway during wing
development remains unclear. To investigate whether dally indeed
affects Dpp signaling activity in the developing wing, we examined the effect
of dally misexpression on pMad levels. We found that the pMad signal
was significantly elevated in dally-overexpressing clones induced by
the FLP-OUT system (Fig. 4).
From the observation of 38 clones, this effect of dally+
expression on pMad levels was found to be strictly cell autonomous. This is
consistent with the idea that Dally serves as a co-receptor for Dpp, which
facilitates the assembly of a signaling complex on the cell surface. Given
that Dpp is an unstable molecule with a short half-life
(Teleman and Cohen, 2000
), it
is also likely that Dally stabilizes Dpp or the Dpp/receptor complex by
protecting them from extracellular degrading enzymes, or by reducing the rate
at which the internalized signaling complexes are delivered to lysosomes.
Increased pMad staining was observed also in several clones induced at the
hinge region (Fig. 4D-F).
However, in some clones located in the peripheral wing pouch, no increase of
pMad staining was seen (Fig.
4G-I). As Dpp levels are low and the levels of endogenous
dally expression are already high in these regions, the elevated
levels of Dally in this experiment might not be sufficient to induce pMad
accumulation. Thus, dally positively controls Dpp signaling in a cell
autonomous fashion, and the sensitivity of cells to dally
misexpression is different depending on the position of the misexpressing
cells in the wing pouch.

View larger version (147K):
[in this window]
[in a new window]
|
Fig. 4. Ectopic expression of dally increases the sensitivity of cells to
Dpp. Three examples are shown for anti-pMad antibody staining of wing discs
bearing clones of cells overexpressing dally. (A,D,G) Patterns of
pMad. (B,E,H) Position of dally-expressing clones marked by GFP
expression. (C,F,I) Overlay images. The pMad levels are increased in the
dally-expressing clones in the wing pouch (arrows) and the hinge
region (arrowheads). Clones induced in the peripheral domain did not show the
elevated pMad signals (asterisks).
|
|
dally affects the Dpp morphogen activity gradient
formation
We next tested whether dally mutations affect the spatial
patterning of Dpp activity by using an anti-pMad antibody. As is evident from
Fig. 5, pMad patterns in wing
discs from dally-hypomorphic mutants
(dallygem/dallygem and
dallygem/dallyDP-527) show
two abnormal features. First, the mutant discs lose the ability to
downregulate Dpp signaling in A/P border cells. Second, in the receiving
cells, the smooth gradient of the pMad signal was not seen in the mutant
discs. This was particularly evident in the P compartment; the pMad levels are
high at the central domain (two-cell widths from the compartment boundary) but
suddenly fall in sites where normally they would decline gradually. As a
result, dally mutant discs show abnormally high levels of pMad in the
center of the disc and low pMad levels in the peripheral regions
(Fig. 5C,G). We also observed a
similar but less severe phenotype in dally heterozygotes
(Fig. 5B,F), which suggests
that gradient formation seems to be sensitive to dally gene
dosage.

View larger version (88K):
[in this window]
[in a new window]
|
Fig. 5. dally affects the Dpp morphogen activity gradient. (A-D) Anti-pMad
antibody staining of the wing discs from wild-type (A),
dallygem/+ (B),
dallygem/dallygem (C) and
dpp-GAL4/UAS-dally (D) animals. Arrowheads indicate the AP boundary.
(E-H) Graphs indicate intensity profiles for pMad levels shown in A-D.
|
|
We found that overexpression of dally at the A/P border cells also
results in abnormal pMad distribution (Fig.
5D,H). The pMad levels in these discs were high at the central
region but abnormally low in the receiving cells. Unlike the pMad patterns
observed in dally mutants, the high pMad signals were restricted to
the dally-overexpressing domain and were not seen in the receiving
cells. This pattern suggests that excess Dally sequesters Dpp proteins at the
site of expression.
We noticed that the pMad pattern seen at the dally mutants is
similar to the abnormality observed in discs overexpressing tkv, in
which the elevated levels of Tkv retard the distribution of Dpp protein (data
not shown) (Lecuit and Cohen,
1998
; Tanimoto et al.,
2000
). This similarity between dally mutants and
tkv-overexpressing discs raises the possibility that dally
normally downregulates tkv expression. In this scenario,
dally mutations would induce high levels of tkv expression,
resulting in abnormal distribution of pMad signals. To determine if this is
the case, we analyzed tkv expression in the same
dally-mutant backgrounds that had been used for the analysis of the
pMad distribution (dallygem/dallygem and
dallygem/dallyDP-527). We
found that levels and expression patterns of tkv::lacZ
(Fig. 6A,B) and Tkv protein
(Fig. 6C,D) in dally
mutants were indistinguishable from those in wild-type discs. This result
indicates that Dally regulates Dpp activity gradient formation via a mechanism
that is independent of tkv expression.

View larger version (176K):
[in this window]
[in a new window]
|
Fig. 6. Expression of tkv in dally mutant wing disc. (A,B)
tkv-enhancer trap expression was monitored using
anti-ß-galactosidase antibody staining in wild-type (A) and
dallygem/dallygem (B) backgrounds. (C,D)
Anti-Tkv antibody staining of wild-type (C) and
dallygem/dallygem (D) wing discs. tkv
expression is not affected by the combinations of dally hypomorphic
alleles, dallygem/dallygem and
dallygem/dallyDP-527 (data
not shown), that alter the shape of the Dpp activity gradient
(Fig. 5C).
|
|
dally affects the Dpp ligand gradient formation
To examine the effect of dally mutations on the distribution of
Dpp morphogen, we expressed Dpp-GFP in the region where it is endogenously
expressed using dpp-GAL4. In wild-type discs, Dpp-GFP was detectable
as intracellular punctate spots and on the surface of the receiving cells
(Fig. 7A), as previously
reported (Entchev et al., 2000
;
Teleman and Cohen, 2000
).
Dpp-GFP migrates throughout the wing pouch region, forming a shallow but
evident gradient. However, in dally-mutant discs, we could not detect
an evident gradient of Dpp distribution in the receiving cells
(Fig. 7B-D). In general, mutant
discs showed a lower level of cell surface signals, suggesting reduced
stability of Dpp.

View larger version (135K):
[in this window]
[in a new window]
|
Fig. 7. Distribution of Dpp-GFP in dally mutant wing disc. Dpp-GFP was
expressed in A/P border cells by dpp-GAL4 and its distribution was
monitored. Wild-type for dally gene (A),
dallygem/dallygem (B-D), and
dpp-GAL4/UAS-dally (E) larvae. All discs were heterozygous for
dppd12 (see Materials and Methods). (F) Intensity profiles
for Dpp-GFP in the posterior half of discs with three different genotypes are
shown. 10, 8 and 12 samples were used for wild-type (green), dally
homozygote (red) and dpp-GAL4/UAS-dally (blue), respectively, to
obtain the averaged profiles.
|
|
To determine whether dally overexpression at the A/P border cells,
which causes abnormal patterns of pMad
(Fig. 5D,H), also affects Dpp
ligand gradient formation, we observed Dpp-GFP distribution in discs where
dally was co-expressed with Dpp-GFP using dpp-GAL4.
Consistent with the pMad patterns (Fig.
5D), Dpp was restricted to the dally-overexpressing
region and failed to migrate properly (Fig.
7E). This suggests that Dally binds to Dpp protein and limits its
distribution. Intensity profiles of these discs (compared in
Fig. 7F) show that both
reduction of dally and overexpression of dally at the A/P
border cells result in a shallower gradient and lower levels of Dpp in the
receiving cells. Taken together, Dally regulates formation of both Dpp ligand
and activity gradients. In addition, our results strongly suggest that Dally
plays at least two roles in the formation of the Dpp signaling gradient: (1)
it regulates the sensitivity of cells to Dpp in a cell autonomous fashion; and
(2) it affects Dpp protein distribution, which is a non-autonomous effect.
 |
DISCUSSION
|
---|
Mechanisms of Dally function in Dpp signaling
Although Dpp is one of the most extensively studied morphogens, the
molecular mechanisms by which the Dpp morphogen gradient is generated and
maintained are poorly understood. Previously, it has been suggested that HSPGs
affect signaling and distribution of BMPs
(Grisaru et al., 2001
;
Jackson et al., 1997
;
Paine-Saunders et al., 2002
;
Paine-Saunders et al., 2000
).
The present study demonstrates that dally controls shape of both the
ligand and the activity gradients of Dpp in the developing wing. How does
dally contribute to the Dpp gradient formation? In vitro analyses
using mammalian tissue culture cells have established that HSPGs can increase
FGF signaling by stabilizing FGF/FGF receptor complexes
(Sperinde and Nugent, 1998
;
Sperinde and Nugent, 2000
).
Several lines of evidence indicated that the dosage of HSPGs is an important
factor for FGF signaling. For example, sodium chlorate treatment, which
inhibits the sulfation of heparan sulfate, reduces the biological response of
cells to FGF; the response can be restored by an exogenous supplement of
heparin. However, restoration is seen only at an optimal concentration of
heparin; excess heparin competes for FGF with signaling complex, resulting in
a reduction of signaling (Krufka et al.,
1996
). In the Drosophila wing, ectopic expression of
Dally-like, another glypican related to Dally, leads to a massive accumulation
of extracellular Wg protein and compromises Wg signal transduction, suggesting
that the glypicans can affect ligand stability and distribution
(Baeg et al., 2001
).
On the basis of these previous studies as well as our data, Dally would
appear to have both positive and negative roles on Dpp signaling
(Fig. 8A). In its positive
role, Dally serves as a co-receptor for Dpp, stabilizing Dpp protein and
enhancing signaling. Conversely, given that Dpp is a heparin-binding protein
(Groppe et al., 1998
), Dally
may bind Dpp through its heparan sulfate chains and reduce the amount of free
Dpp ligands. Thus, Dally affects the Dpp gradient at two distinct steps:
signal transduction (autonomous effect) and ligand distribution
(non-autonomous effect). We propose a model in which alterations in the shapes
of the Dpp ligand and the activity gradients caused by dally
mutations and dally overexpression are interpreted as sum of these
plus and minus effects of Dally function. In this model, Dally normally
sequesters Dpp protein to some extent in A/P border cells, where
dally levels are very high. Therefore, reduced levels of Dally in
mutant discs may result in the release of Dpp ligand and, consequently, higher
levels of signaling activity in the central region. Therefore, dally
mutations may severely reduce the stability of Dpp protein as well as its
signaling activity in the receiving cells. When dally is
overexpressed in A/P border cells, Dpp is trapped by binding to excess Dally
and fails to distribute properly.

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 8. Expression patterns and function of dally contribute to shaping
the Dpp morphogen gradient in wing disc. (A) Model for Dally function in Dpp
signaling. Dally (red) forms a signaling complex with Dpp (green) and receptor
molecules (yellow) on the cell surface (left). Purple arrows represent
signaling activity. Increased levels of Dally can enhance Dpp signaling by
stabilizing the signaling complex (middle). However, excess levels of Dally
sequester Dpp protein and show an inhibitory effect on signaling (right). (B)
Levels of tkv expression (yellow), dally expression (red)
and Dpp signaling (purple). Expression of both Tkv (a Dpp receptor) and Dally
(a Dpp co-receptor) is regulated by several common molecular pathways in the
wing. (1) Hh signaling suppresses tkv and activates dally.
(2) En induces tkv and represses dally. (3) Dpp signaling
downregulates both genes. As the Tkv receptor and Dally co-receptor mediate
Dpp signaling, this regulatory pathway forms a negative-feedback loop. At
anterior and posterior edges of the wing pouch, lower levels of Dpp signaling
result in high levels of Tkv and Dally, which sensitize cells to Dpp.
|
|
The model described above is based on the idea that the Dpp gradient is
established by diffusion. The diffusive mechanisms of morphogen gradient
formation are supported by a recent theoretical analysis
(Lander et al., 2002
).
However, our results do not rule out the possibility that Dally plays a more
active role in facilitating Dpp diffusion or `carries' Dpp protein. For
example, it is possible that Dally is required for the Dpp movement through
the transcytosis pathway (Entchev et al.,
2000
; Gonzalez-Gaitan and
Jackle, 1999
) or other transport systems, such as cytonemes
(Ramirez-Weber and Kornberg,
1999
) and argosomes (Greco et
al., 2001
).
Regulated expression of receptor and co-receptor for stable morphogen
gradient
We also showed that dally expression is regulated by the same set
of signaling pathways that control expression of tkv. Both genes are
regulated by Hh in A/P border cells and by En in the P compartment
(Fig. 1,
Fig. 8B), but the effects of Hh
and En on dally are opposite to those on tkv. In addition,
dally expression is negatively controlled by Dpp signaling. Through
this mechanism, relative levels of dally expression are higher at the
anterior and posterior distal edges. Therefore, dally and
tkv show similar patterns of expression with one exception: the level
of dally expression is high in A/P border cells, where Dpp is
synthesized and secreted, but by contrast, tkv expression levels are
low in this region (Fig. 1,
Fig. 8B). The high levels of
dally in the peripheral regions could sensitize cells to low levels
of Dpp, as has been shown for tkv
(Lecuit and Cohen, 1998
).
These regulatory pathways appear to form negative feedback loops, which may
stabilize the shape of the Dpp morphogen gradient. Thus, the regulated
expression and function of Dally are crucial factors in the generation and
maintenance of the Dpp morphogen gradient.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to S. Cohen, M. Nakamura, T. Tabata, P. ten Dijke and the
Bloomington Stock Center for antibodies and fly stocks. We thank S. Selleck,
S. Stringer and W. Waldrip for helpful discussions. This work was supported in
part by the Human Frontier Science Program.
 |
Footnotes
|
---|
* These authors contributed equally to this work 
 |
REFERENCES
|
---|
Alcedo, J., Ayzenzon, M., von Ohlen, T., Noll, M. and Hooper, J.
E. (1996). The Drosophila smoothened gene encodes a
seven-pass membrane protein, a putative receptor for the hedgehog signal.
Cell 86,221
-232.[Medline]
Baeg, G. H., Lin, X., Khare, N., Baumgartner, S. and Perrimon,
N. (2001). Heparan sulfate proteoglycans are critical for the
organization of the extracellular distribution of Wingless.
Development 128,87
-94.[Abstract/Free Full Text]
Bellaiche, Y., The, I. and Perrimon, N. (1998).
Tout-velu is a Drosophila homologue of the putative tumour suppressor EXT-1
and is needed for Hh diffusion. Nature
394, 85-88.[CrossRef][Medline]
Burke, R. and Basler, K. (1996). Dpp receptors
are autonomously required for cell proliferation in the entire developing
Drosophila wing. Development
122,2261
-2269.[Abstract/Free Full Text]
Entchev, E. V., Schwabedissen, A. and Gonzalez-Gaitan, M.
(2000). Gradient formation of the TGF-beta homolog Dpp.
Cell 103,981
-991.[Medline]
Fujise, M., Izumi, S., Selleck, S. B. and Nakato, H.
(2001). Regulation of dally, an integral membrane proteoglycan,
and its function during adult sensory organ formation of Drosophila.
Dev. Biol. 235,433
-448.[CrossRef][Medline]
Funakoshi, Y., Minami, M. and Tabata, T.
(2001). mtv shapes the activity gradient of the Dpp morphogen
through regulation of thickveins. Development
128, 67-74.[Abstract/Free Full Text]
Gerlitz, O. and Basler, K. (2002). Wingful, an
extracellular feedback inhibitor of Wingless. Genes
Dev. 16,1055
-1059.[Abstract/Free Full Text]
Giraldez, A. J., Copley, R. R. and Cohen, S. M.
(2002). HSPG modification by the secreted enzyme Notum shapes the
Wingless morphogen gradient. Dev. Cell
2, 667-676.[Medline]
Golic, K. G. (1991). Site-specific
recombination between homologous chromosomes in Drosophila.
Science 252,958
-961.[Medline]
Gonzalez-Gaitan, M. and Jackle, H. (1999). The
range of spalt-activating Dpp signalling is reduced in endocytosis-defective
Drosophila wing discs. Mech. Dev.
87,143
-151.[CrossRef][Medline]
Greco, V., Hannus, M. and Eaton, S. (2001).
Argosomes: a potential vehicle for the spread of morphogens through epithelia.
Cell 106,633
-645.[Medline]
Grisaru, S., Cano-Gauci, D., Tee, J., Filmus, J. and Rosenblum,
N. D. (2001). Glypican-3 modulates BMP- and FGF-mediated
effects during renal branching morphogenesis. Dev.
Biol. 231,31
-46.[CrossRef][Medline]
Groppe, J., Rumpel, K., Economides, A. N., Stahl, N., Sebald, W.
and Affolter, M. (1998). Biochemical and biophysical
characterization of refolded Drosophila DPP, a homolog of bone morphogenetic
proteins 2 and 4. J. Biol. Chem.
273,29052
-29065.[Abstract/Free Full Text]
Ito, K., Awano, W., Suzuki, K., Hiromi, Y. and Yamamoto, D.
(1997). The Drosophila mushroom body is a quadruple structure of
clonal units each of which contains a virtually identical set of neurones and
glial cells. Development
124,761
-771.[Abstract/Free Full Text]
Jackson, S. M., Nakato, H., Sugiura, M., Jannuzi, A., Oakes, R.,
Kaluza, V., Golden, C. and Selleck, S. B. (1997). Dally, a
Drosophila glypican, controls cellular responses to the TGF-beta-related
morphogen, Dpp. Development
124,4113
-4120.[Abstract/Free Full Text]
Krufka, A., Guimond, S. and Rapraeger, A. C.
(1996). Two hierarchies of FGF-2 signaling in heparin: mitogenic
stimulation and high-affinity binding/receptor transphosphorylation.
Biochemistry 35,11131
-11141.[CrossRef][Medline]
Lander, A. D., Nie, Q. and Wan, F. Y. (2002).
Do morphogen gradients arise by diffusion? Dev. Cell
2, 785-796.[Medline]
Lecuit, T. and Cohen, S. M. (1998). Dpp
receptor levels contribute to shaping the Dpp morphogen gradient in the
Drosophila wing imaginal disc. Development
125,4901
-4907.[Abstract/Free Full Text]
Lecuit, T., Brook, W. J., Ng, M., Calleja, M., Sun, H. and
Cohen, S. M. (1996). Two distinct mechanisms for long-range
patterning by Decapentaplegic in the Drosophila wing.
Nature 381,387
-393.[CrossRef][Medline]
Lin, X. and Perrimon, N. (1999). Dally
cooperates with Drosophila Frizzled 2 to transduce Wingless signalling.
Nature 400,281
-284.[CrossRef][Medline]
Morimura, S., Maves, L., Chen, Y. and Hoffmann, F. M.
(1996). Decapentaplegic overexpression affects Drosophila wing
and leg imaginal disc development and wingless expression. Dev.
Biol. 177,136
-151.[CrossRef][Medline]
Nakato, H., Futch, T. A. and Selleck, S. B.
(1995). The division abnormally delayed (dally) gene: a putative
integral membrane proteoglycan required for cell division patterning during
postembryonic development of the nervous system in Drosophila.
Development 121,3687
-3702.[Abstract/Free Full Text]
Nellen, D., Affolter, M. and Basler, K. (1994).
Receptor serine/threonine kinases implicated in the control of Drosophila body
pattern by decapentaplegic. Cell
78,225
-237.[Medline]
Nellen, D., Burke, R., Struhl, G. and Basler, K.
(1996). Direct and long-range action of a DPP morphogen gradient.
Cell 85,357
-368.[Medline]
Paine-Saunders, S., Viviano, B. L., Zupicich, J., Skarnes, W. C.
and Saunders, S. (2000). Glypican-3 controls cellular
responses to Bmp4 in limb patterning and skeletal development. Dev.
Biol. 225,179
-187.[CrossRef][Medline]
Paine-Saunders, S., Viviano, B. L., Economides, A. N. and
Saunders, S. (2002). Heparan sulfate proteoglycans retain
Noggin at the cell surface: a potential mechanism for shaping bone
morphogenetic protein gradients. J. Biol. Chem.
277,2089
-2096.[Abstract/Free Full Text]
Persson, U., Izumi, H., Souchelnytskyi, S., Itoh, S., Grimsby,
S., Engstrom, U., Heldin, C. H., Funa, K. and ten Dijke, P.
(1998). The L45 loop in type I receptors for TGF-beta family
members is a critical determinant in specifying Smad isoform activation.
FEBS Lett. 434,83
-87.[CrossRef][Medline]
Ramirez-Weber, F. A. and Kornberg, T. B.
(1999). Cytonemes: cellular processes that project to the
principal signaling center in Drosophila imaginal discs.
Cell 97,599
-607.[Medline]
Singer, M. A., Penton, A., Twombly, V., Hoffmann, F. M. and
Gelbart, W. M. (1997). Signaling through both type I DPP
receptors is required for anterior-posterior patterning of the entire
Drosophila wing. Development
124, 79-89.[Abstract/Free Full Text]
Sperinde, G. V. and Nugent, M. A. (1998).
Heparan sulfate proteoglycans control intracellular processing of bFGF in
vascular smooth muscle cells. Biochemistry
37,13153
-13164.[CrossRef][Medline]
Sperinde, G. V. and Nugent, M. A. (2000).
Mechanisms of fibroblast growth factor 2 intracellular processing: a kinetic
analysis of the role of heparan sulfate proteoglycans.
Biochemistry 39,3788
-3796.[CrossRef][Medline]
Strigini, M. and Cohen, S. M. (1997). A
Hedgehog activity gradient contributes to AP axial patterning of the
Drosophila wing. Development
124,4697
-4705.[Abstract/Free Full Text]
Struhl, G. and Basler, K. (1993). Organizing
activity of wingless protein in Drosophila. Cell
72,527
-540.[Medline]
Tabata, T., Schwartz, C., Gustavson, E., Ali, Z. and Kornberg,
T. B. (1995). Creating a Drosophila wing de novo, the role of
engrailed, and the compartment border hypothesis.
Development 121,3359
-3369.[Abstract/Free Full Text]
Tanimoto, H., Itoh, S., ten Dijke, P. and Tabata, T.
(2000). Hedgehog creates a gradient of DPP activity in Drosophila
wing imaginal discs. Mol. Cell
5, 59-71.[Medline]
Teleman, A. A. and Cohen, S. M. (2000). Dpp
gradient formation in the Drosophila wing imaginal disc.
Cell 103,971
-980.[Medline]
The, I., Bellaiche, Y. and Perrimon, N. (1999).
Hedgehog movement is regulated through tout velu-dependent synthesis of a
heparan sulfate proteoglycan. Mol. Cell
4, 633-639.[Medline]
Toyoda, H., Kinoshita-Toyoda, A. and Selleck, S. B.
(2000). Structural analysis of glycosaminoglycans in Drosophila
and Caenorhabditis elegans and demonstration that tout-velu, a Drosophila gene
related to EXT tumor suppressors, affects heparan sulfate in vivo.
J. Biol. Chem. 275,2269
-2275.[Abstract/Free Full Text]
Tsuda, M., Kamimura, K., Nakato, H., Archer, M., Staatz, W.,
Fox, B., Humphrey, M., Olson, S., Futch, T., Kaluza, V. et al.
(1999). The cell-surface proteoglycan Dally regulates Wingless
signalling in Drosophila. Nature
400,276
-280.[CrossRef][Medline]
Wieser, R., Wrana, J. L. and Massague, J.
(1995). GS domain mutations that constitutively activate T beta
R-I, the downstream signaling component in the TGF-beta receptor complex.
EMBO J. 14,2199
-2208.[Abstract]
Xu, T. and Rubin, G. M. (1993). Analysis of
genetic mosaics in developing and adult Drosophila tissues.
Development 117,1223
-1237.[Abstract/Free Full Text]
Related articles in Development:
- Dallying with Dpp morphogen gradients
Development 2003 130: 801.
[Full Text]