Division of Developmental Biology, Children's Hospital Medical Center, Cincinnati, OH 45229, USA
Author for correspondence (e-mail:
linyby{at}chmcc.org)
Accepted 30 October 2003
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SUMMARY |
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Key words: Hedgehog, Heparan sulphate proteoglycans, Glypican, Dally; Dally-like, Drosophila, Morphogen gradient formation, Signaling
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Introduction |
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Hh signalling is essential for many developmental processes in
Drosophila (Ingham and McMahon,
2001). The role of Hh is particularly well documented during wing
disc development. In the wing disc, Hh is expressed in the posterior (P)
compartment and moves anteriorly to transduce its signal in a narrow stripe of
tissue in the anterior compartment (A) abutting the anteroposterior (AP)
boundary. Hh signalling induces the expression of its target genes including
decapentaplegic (dpp), patched (ptc),
collier (col) and engrailed (en) in a
concentration-dependant manner (Chen and
Struhl, 1996
; Mullor et al.,
1997
; Strigini and Cohen,
1997
; Vervoort et al.,
1999
; Zecca et al.,
1995
). Although Dpp diffuses bidirectionally into both
compartments and functions as a long-range morphogen to control the growth and
patterning of cells in the entire wing
(Lecuit et al., 1996
;
Nellen et al., 1996
), Hh
signalling is required for the proper patterning of the region near the AP
compartment boundary (Chen and Struhl,
1996
; Strigini and Cohen,
1997
; Vervoort et al.,
1999
). Genetic screens in Drosophila have identified
three components including Patched (Ptc), Dispatched (Disp) and Tout-velu
(Ttv), which play distinct roles in Hh distribution. Disp and Ptc are
multi-span transmembrane proteins and essential for Hh trafficking in its
producing cells and receiving cells, respectively. Disp is required in
Hh-producing cells and it appears to control the release of Hh-Np from the
producing cells (Burke et al.,
1999
). In the absence of Disp function, Hh-Np accumulates in its
producing cells and fails to move into anterior receiving cells
(Burke et al., 1999
). The
distribution of Hh in its receiving cells is regulated by its receptor Ptc
that is expressed in all A compartment cells and is upregulated by Hh
signalling (Forbes et al.,
1993
; Goodrich et al.,
1996
; Marigo et al.,
1996
). Upregulated Ptc protein in the Hh-responding wing cells
sequesters Hh and thereby restricts the further movement of Hh into the A
compartment (Chen and Struhl,
1996
).
Genetic studies of the function of Ttv in Drosophila have
demonstrated an essential role of heparan sulphate proteoglycan(s)(HSPG) in Hh
movement in its receiving cells (Bellaiche
et al., 1998; The et al.,
1999
). ttv is a Drosophila homolog of the
mammalian EXT1 tumor suppressor gene
(McCormick et al., 1998
)
encoding a heparan sulphate copolymerase
(Lind et al., 1998
;
The et al., 1999
). HSPGs are
cell surface macromolecules consisting of a protein core and a number of
heparan sulphate glycosaminoglycan (HS GAG) chains
(Bernfield et al., 1999
;
Lin and Perrimon, 2000
;
Perrimon and Bernfield, 2000
).
In Drosophila, there are two glypicans, division abnormally
delayed (dally) (Nakato et
al., 1995
) and dally-like (dly)
(Baeg et al., 2001
;
Khare and Baumgartner, 2000
),
one Drosophila syndecan (dsyndecan)
(Spring et al., 1994
), and one
perlecan encoded by terribly reduced optic lobes
(trol) (Park et al.,
2003
; Voigt et al.,
2002
). Glypicans represent one of main cell surface HSPGs, which
linked to the plasma membrane by a glycosyl phosphatidylinositol (GPI) linker
(Bernfield et al., 1999
;
Lin and Perrimon, 2000
;
Perrimon and Bernfield, 2000
).
Previous studies have demonstrated that the movement of Hh from P to A
compartment is defective in cells mutant for tout-velu (ttv)
(Bellaiche et al., 1998
).
Cholesterol-unmodified Hh-N, but not Hh-Np, is independent of Ttv function for
its movement (The et al.,
1999
). A recent study further showed that cholesterol in Hh-Np is
required for the formation of large punctate structures in embryonic
epidermis, whereas the movement of these large punctate structures across
cells is contingent upon the activity of Ttv
(Gallet et al., 2003
).
Together, these studies suggest that the proteoglycan(s) modified by Ttv is
specifically required for the movement of cholesterol-modified Hh-Np. However,
the precise function of HSPG(s) in regulating Hh movement and its signalling
is not understood. In particular, it is currently unknown which class of HSPGs
is involved in Hh signalling.
In this report, we provide evidence that Dally and Dly, two glypican members of HSPGs, are the substrates for Ttv and are involved in Hh movement and its subsequent signalling. We show that Dly is required for Hh signalling during embryogenesis, and that both Dally and Dly are required and redundant in Hh movement during wing disc development. Importantly, we found that a narrow strip of cells defective in HS GAG biosynthesis or mutant for dally-dly severely impaired the further movement of Hh to more anterior cells in the wing disc, suggesting that HSPG-mediated Hh movement through a field of cells is regulated by a cell-to-cell mechanism rather than by free diffusion. We further demonstrate that Hh movement is independent of dynamin-mediated endocytosis. These findings led us to propose that the glypicans Dally and Dly transfer Hh along the cell membrane to pattern a field of cells.
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Materials and methods |
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Generation of dally and dly null alleles
dally80 was generated by P-element mediated mutagenesis
using dallyP1 (Nakato
et al., 1995). dally80 contains a deletion
from 724 to +415 (A in the ATG start codon is designated as 1) that
covers the ATG start codon followed by 54 amino acids including the signal
peptide as well as part of the first intron. dlyA187 is an
EMS-induced mutant that was isolated from a deficiency screen using
Df(3L)fz-M21. dlyA187 contains a deletion of 26
nucleotides resulting in a reading frame shift from amino acid 205.
dlyA187 lacks part of the cysteine-rich region, the entire
GAG attachment domain and the glycosylphosphatidylinositol (GPI)-anchoring
signal (Lin and Perrimon,
2000
; Perrimon and Bernfield,
2000
).
Wing phenotype analysis
Wing phenotypes were analyzed by `directed mosaic'
(Duffy et al., 1998) using
UAS-Flipase(flp)/vgQ1206-Gal4 as described
(Belenkaya et al., 2002
). Males
of genotype w, dally80 (or dlyA187 or
dally80-dlyA187) FRT2A / TM6B were
crossed with females of genotype of w; vg Q1206-Gal4 UAS-flp;
FRT2A / TM3 that express flp primarily in the wing
imaginal disc cells under vg Q1206-Gal4 control. This flp
activity mediates a high frequency of mitotic recombination, generating clones
of cells homozygous for dally80 or dlyA187 or
dally80-dlyA187.
Generation of germline clones and marked wing clones
dly germline clones were generated by the autosomal FLP-DFS
technique (Chou and Perrimon,
1996). Clones of mutant cells 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 2 hours. To generate shibire mutant clones, larvae were
allowed to grow at 18°C and were shifted to 32°C for 5 or 10 hours
prior to fixation and antibody staining. The genotypes used in our analyses
were as follows.
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Results |
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Our previous study showed that disruption of dly in embryos by RNA
interference (RNAi) led to a strong segment polarity defect, suggesting that
Dly is likely to be involved in Hh and/or Wingless (Wg) signalling in
embryonic epidermis (Baeg et al.,
2001). To explore the potential role of Dly in Hh signalling, we
isolated a number of dly mutant alleles using EMS mutagenesis.
dlyA187 is a null allele (see Materials and methods for
details) and is used for further analyses. Animals zygotically mutant for
dly appears to have normal cuticle patterning (data not shown) and
survive until third instar larvae. However, homozygous mutant embryos derived
from females lacking maternal dly activity (referred to as
dly embryos hereafter) die with a strong segment-polarity phenotype
(Fig. 1C) resembling those of
mutants of the segment polarity genes hh
(Fig. 1B) and wg (data
not shown). In dly embryos, both En expression and wg
transcription fade by stage 10 (Fig.
1E,G), suggesting further that dly is involved in the Hh
and/or Wg pathways.
To further determine whether Dly activity is required for Hh signalling in
embryogenesis, we examined Hh signalling activity in dly embryos
during mesoderm development. Hh and Wg signalling have distinct roles in
patterning embryonic mesoderm. Hh signalling activates the expression of a
mesodermal specific gene bagpipe (bap) in the anterior
region of each parasegment, whereas Wg signalling inhibits bap
expression in the posterior region (Azpiazu
et al., 1996). bap expression is diminished in the
hh mutant, but is expanded to the posterior parasegment in the
wg mutant (Azpiazu et al.,
1996
). Consistent with a role of Dly in Hh signalling, we found
that bap expression was strikingly reduced in dly embryos
(Fig. 1I). Together with the
segment polarity phenotype, these results strongly argue that Dly is required
for Hh signalling during embryogenesis.
dally and dly are required for Hh signalling in wing development
We further examined the role of Dly in Hh signalling during wing
development in which Hh and Wg signalling function independently of each
other. In the wing disc, Hh signalling induces the expression of its target
genes in a narrow stripe of tissue in the A compartment abutting the AP
boundary. Hh signalling patterns the central domain of wing blade and controls
the positioning of longitudinal veins L3 and L4
(Mullor et al., 1997;
Slusarski et al., 1995
;
Strigini and Cohen, 1997
). We
first examined the roles of Dly in Hh signalling by analyzing adult wing
defects using `directed mosaic' technique
(Belenkaya et al., 2002
;
Duffy et al., 1998
) (see
Materials and methods for details). To our surprise, we did not observe any
detectable phenotypes in adult wings bearing dly mutant clones (data
not shown). We reasoned that Hh signalling may be mediated by other HSPGs in
the wing. One candidate is the glypican dally
(Nakato et al., 1995
) that has
previously been shown to be involved in Wg
(Lin and Perrimon, 1999
;
Tsuda et al., 1999
) and Dpp
signalling (Jackson et al.,
1997
). Because available dally alleles used previously
were hypomorphic, we generated several dally null alleles by
P-element mediated mutagenesis. dally80 is a null allele
and was used for our analysis (see Materials and methods for details).
However, similar to other dally alleles, homozygous
dally80animals are viable. The wing bearing
dally80 clones exhibits a partial loss of the L5 vein with
a high penetrance, but no detectable defects in the central domain of wing
blade (arrow in Fig. 2B). To
determine whether dally and dly have overlapping roles in Hh
signalling in wing development, we generated clones mutant for both
dally80 and dlyA187 (referred as
dally-dly hereafter). Interestingly, the adult wings bearing clones
mutant for dally-dly show L3-L4 fusion (arrow in
Fig. 2C). This phenotype is
typical of loss of Hh function, suggesting that Dally and Dly play redundant
roles in Hh signalling in wing development.
We further examined the effect of dally-dly on Hh signalling by
directly analyzing the expression of Hh target genes including ptc,
en and col. The following experiments provide compelling
evidence for the involvement of Dally and Dly in Hh signalling in the wing
disc. First, in the wild-type wing disc, Ptc expression is normally elevated
by Hh signalling in anterior cells adjacent to AP boundary
(Fig. 2D). Levels of Ptc
expression are reduced in dally-dly clones
(Fig. 2E,E',F,F').
Second, En expression is induced by Hh signalling over three to four cell
diameters in anterior cells adjacent to the AP boundary
(Fig. 2G)
(Blair, 1992). This induction
is eliminated in dally-dly clones
(Fig. 2H,H'). Third, Col
is normally expressed in four cell diameters adjacent to the AP boundary
(Fig. 2I)
(Vervoort et al., 1999
). In
the dally-dly clone, Col expression is reduced and limited to two
rows of cells at the posterior edge of the clone and with a lower level in the
more anterior row (Fig.
2J,J'). Collectively, these observations argue that Dally
and Dly are required and are redundant in Hh signalling in the wing disc.
Ttv affects HS GAG modifications in Dally and Dly
Previous studies have demonstrated the involvement of Ttv in Hh signalling
(Bellaiche et al., 1998;
The et al., 1999
).
ttv is a Drosophila homolog of the mammalian EXT1
gene encoding the HS co-polymerase required for the biosynthesis of HS GAG
chains (Lind et al., 1998
;
The et al., 1999
). We reasoned
that if Dally and Dly are involved in Hh signalling, they may be modified by
Ttv. We therefore examined the modification of Dally and Dly in ttv
mutant larvae by western blot analysis. As shown previously
(Baeg et al., 2001
;
Giraldez et al., 2002
;
Lin and Perrimon, 1999
;
Tsuda et al., 1999
), in
wild-type larval extracts, both Dally and Dly migrate as bands with high
molecular masses, characteristic of HSPGs to which negatively charged HS GAG
chains are attached (Fig.
3A,B). In homozygous ttv larvae, the high molecular mass
of Dly is shifted to a relatively lower molecular mass. The high molecular
mass of Dally is also significantly reduced. These results indicate that both
Dally and Dly are probably the substrates of Ttv.
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Hh movement is independent of dynamin-mediated endocytosis
The cell-to-cell movement of Hh could be mediated by endocytic routes
through planar transcytosis (Bejsovec and
Wieschaus, 1995; Moline et
al., 1999
; Seto et al.,
2002
) or by movement along the cell surface. To determine whether
Hh movement is mediated by planar transcytosis, we examined Hh distribution
and its subsequent signalling in clones mutant for
shibirets1 (shits1), which encodes the
Drosophila homolog of the GTPase dynamin required for endocytosis
(Chen et al., 1991
;
van der Bliek and Meyerowitz,
1991
).
First, we examined the distribution of Hh in shits1 clones that were allowed to grow at permissive temperature (18°C) and then shifted to 32°C for 5 hours to inactivate Shibire. Hh movement in its receiving cells mutant for shits1 is not defective under these conditions (Fig. 5B). Hh is not accumulated in front of the shits1 clone facing posterior Hh-producing cells, which is predicted if the shits1 clone blocks the cell-to-cell movement of Hh protein (Fig. 5B,B'). However, two observations suggest that the internalization of Hh is indeed blocked in shits1 mutant cells. First, Hh is accumulated on the cell membrane of Hh receiving cells within shits1 clones. This accumulation of Hh is prominent in basal (Fig. 5C) and apical (data not shown) regions of the columnar cells. Second, we observed a dramatic reduction in the number of large punctate particles in shits1 clones (Fig. 5B), which are presumably internalized vesicles containing both Hh and Ptc, and are readily seen in three to four rows of A cells abutting the AP boundary in wild-type disc (Fig. 5A, arrows).
We further examined the activity of Hh signalling in clones mutant for shits1. Both Ptc and Ci expression levels are not reduced in clones mutant for shits1 (Fig. 5D,D',E,E'), even when the incubation at 32°C was extended to 10 hours (data not shown). Instead, we found that Ptc expression is expanded within the large shits1 clone (Fig. 5D,D'), which is presumably due to the defective internalization and subsequent degradation of the Hh protein. Importantly, Ptc expression in wild-type cells behind (anterior to) the small shits1 clone is still maintained (arrow in Fig. 5D), indicating that Hh is able to pass through shits1 mutant cells. However, we found that the potentiation of Hh signalling caused by Hh accumulation in shits1 mutant cells is mild, as Ci expression pattern in large shits1 clones is barely changed (Fig. 5E,E'). Taken together, these data suggest that dynamin-mediated endocytosis is required for Hh internalization, but neither for its movement nor for its signalling in receiving cells.
HSPGs co-localize with and stabilize Hh
One possible role of HSPGs in Hh movement is to stabilize Hh on the cell
surface by directly interacting and forming complexes with Hh. We tested this
by examining the co-localization of Hh with Dly. We ectopically expressed a
GFP-Dly fusion protein in either the ptc domain or the hh
domain in wing discs. Hh colocalizes with GFP-Dly in both cases as shown by
diffusive membrane staining and punctate particles
(Fig. 6A,B). This finding is
also consistent with our following loss-of-function analyses. We examined Hh
distribution in sfl mutant cells in the wing disc as well as in
dly embryos. Hh staining disappears in sfl mutant clones,
except at a residual level in the posterior-most row of cells
(Fig. 6C). In wild-type
embryos, Hh staining is detected as punctate particles, which are found at
least one cell diameter from its producing cells (En-positive cells)
(Fig. 6D) (Porter et al., 1996a;
Tabata and Kornberg, 1994
;
Taylor et al., 1993
). However,
in dly embryos, these punctate particles disappear and Hh staining
appears to be membrane bound in its producing cells
(Fig. 6E). Similar defects in
Hh distribution were also observed in ttv embryos
(Gallet et al., 2003
;
The et al., 1999
). Together,
these observations suggest that glypicans co-localize with Hh and regulate Hh
movement possibly by forming complex(es) with Hh and stabilizing Hh protein on
the cell surface.
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Discussion |
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Role of Dly in Hh signalling during embryogenesis
This study first demonstrates that Dly is the main HSPG involved in Hh
signalling during embryogenesis, at least in epidermis and mesoderm, the two
tissues that were carefully examined. Three lines of evidence strongly support
our conclusion. First, embryos lacking both maternal and zygotic dly
activities develop a strong segment polarity defect and exhibit diminished
expression of En and Wg (Fig.
1C,E,G). Second, we found that Hh can be detected as punctate
particles at least one cell diameter from its producing cells and these
punctate particles are absent in dly-null embryos
(Fig. 6D,E). Third, we observed
a reduced expression of bap in dly mutant embryos
(Fig. 1I), a phenotype
specifically attributed to the Hh signalling rather than Wg signalling defect.
Previously, it was shown that the punctate particles of Hh staining are absent
in ttv null embryos (The et al.,
1999). Recently, Gallet et al. showed that the formation of such
Hh staining particles, referred as large punctate structures (LPS), requires
cholesterol modification (Gallet et al.,
2003
). They further demonstrated that movement of these large
punctate structures across cells is dependent on Ttv activity
(Gallet et al., 2003
). Our
results are consistent with these observations and suggest that Dly is the
main HSPG involved in the movement of these LPS across cells. It is
conceivable that the punctate particles of Hh staining we observed may
represent Hh-Dly complexes. In this regard, Dly may either prevent secreted Hh
from being degraded and/or facilitate Hh movement from its expression cells to
adjacent receiving cells. These two mechanisms are not mutually exclusive. In
the absence of Dly function, secreted Hh is either degraded or fails to move
to the adjacent cells.
In addition to dly, three other HSPGs, including Dally, Dsyndecan
and Trol, are also expressed in various tissues during embryogenesis. In
particular, dally is expressed in epidermis and has been shown to be
involved in Wg signalling (Lin and
Perrimon, 1999; Toyoda et al.,
2000
). Removal of Dally activity in embryos either by
dally hypomorphic mutants or by RNA interference (RNAi) generates
denticle fusions (Lin and Perrimon,
1999
; Toyoda et al.,
2000
). Further studies demonstrated that the cuticle defect
associated with dally embryos by RNAi is weaker than that of
dly (Baeg et al.,
2001
). Our results in this work suggest that Dly plays more
profound roles in embryonic patterning than Dally. It remains to be determined
whether Dally and other two Drosophila HSPGs are involved in Hh
signalling in other developmental processes during embryogenesis.
Role of Dally and Dly in Hh movement during wing development
We further demonstrate that Dally and Dly are involved and are redundant in
Hh signalling in the wing disc. Consistent with this, we also show that the
GAG chains of Dally and Dly are altered in the absence of Ttv activity,
suggesting that both Dally and Dly are indeed the substrates for Ttv
(Fig. 3A,B). Redundant roles of
cell membrane proteins have been demonstrated in many other signalling
systems. For example, both Frizzled (Fz) and Drosophila Frizzled 2
(Fz2) are redundant receptors for Wg
(Bhanot et al., 1999;
Chen and Struhl, 1999
),
although Fz2 has relative high affinity in binding to Wg protein
(Rulifson et al., 2000
). Dly
protein is distributed throughout the entire wing disc (data not shown).
Previous studies demonstrated that dally is highly expressed at the
AP border (Fujise et al.,
2001
). Interestingly, Dally expression at the AP border is
overlapped with the ptc expression domain and is under the control of
Hh signalling (Fujise et al.,
2001
). It is likely that both Dally and Dly are capable of binding
to Hh and facilitating the movement of the Hh protein. In the absence of one
of them, another member is probably sufficient to facilitate Hh movement.
We notice that dally-dly double mutant clones have relatively weaker defects in Hh signalling in the wing disc than those of the ttv and sfl mutants. One possible explanation is the perdurance of Dally and Dly proteins. Alternatively, two other HSPGs, Dsyndecan and Trol, may also participate in Hh signalling in the absence of Dally and Dly in the wing disc. These issues remain to be examined using both dsyndecan and trol null mutants.
Role of HSPGs in Hh signal transduction
Do HSPGs act as co-receptors in Hh signal transduction? Hh is a
heparin-binding protein and is likely to interact with HSPGs through their HS
GAG chains. In support of this, we show that Dly colocalizes with Hh punctate
particles (Fig. 6A,B). It is
conceivable that Dally and Dly could either transfer Hh to its receptor Ptc or
form a Hh-Dally/Dly-Ptc ternary complex in which Dally and Dly may function to
facilitate Hh-Ptc interaction or stabilize a Hh-Ptc complex. In this regard,
Dally and Dly may function both in transporting Hh protein and acting as
co-receptors in Hh signalling (Lin and
Perrimon, 2003). Consistent with this view, a recent report using
RNAi in tissue culture based assays identified Dly as a new component of the
Hh pathway (Lum et al., 2003
).
It was shown that Dly plays a cell-autonomous role upstream or at the level of
Ptc in activating the expression of Hh responsive-reporter, suggesting a role
of Dly in the delivery of Hh to Ptc.
It is important to note that some of results obtained from tissue culture
based assays (Lum et al.,
2003) are not consistent with our in vivo results reported here as
well as previous studies on Ttv (Bellaiche
et al., 1998
; The et al.,
1999
). Cl-8 cells were originally derived from the wing disc.
However, we found that removal of dly activity alone has no
detectable effect on Hh signalling in the wing disc. This apparent discrepancy
may due to several factors. First, Hh-N, instead of Hh-Np was used as a source
for Hh in their work. Second, Cl-8 cell may have altered the proteoglycan
expression pattern, which can be significantly different from Hh-responding
wing cells in which Dally expression is upregulated by Hh signalling
(Fujise et al., 2001
).
Finally, it is possible that Dly may have a higher capacity than Dally to bind
Hh, as in the case for Wg (Baeg et al.,
2001
). In this regard, removal of Dly will probably lead to more
profound effects than removal of other HSPGs on binding of Hh-N to the cell
surface, perhaps in the delivery of Hh-N to Ptc.
We notice that within sfl, or ttv or dally-dly
mutant clones, the posterior-most cells adjacent to wild-type cells are still
capable of transducing Hh signalling (Fig.
4). It is most likely that Hh proteins bound by Dally and Dly in
wild-type cells can directly interact with Ptc located on the cell surface of
the adjacent mutant cells to transduce its signalling. In support of this
view, Strigini and Cohen previously demonstrated that a Hh-CD2 membrane fusion
protein can activate Hh signalling in its adjacent cells
(Strigini and Cohen, 1997).
Furthermore, studies on Disp have shown that the first row of anterior cells
adjacent to posterior Hh-producing cells have significant Hh signalling
activity in disp mutant wing disc, in which Hh is retained on the
cell surface of its producing cells (Burke
et al., 1999
; Ma et al.,
2002
). Interestingly, we also observed Hh punctate particles in
the posteriormost HSPG mutant cells adjacent to wild-type cells
(Fig. 6C,C'). These Hh
punctate particles are most likely intracellular Hh proteins internalized
through Ptc mediated endocytosis process. In this regard, HSPGs may not be
required for Ptc-mediated Hh internalization.
Mechanism(s) of Glypican-mediated Hh movement
Recent biochemical studies from vertebrate cells have shown that Shh-Np is
secreted from cells and can be readily detected in conditioned culture medium
(Zeng et al., 2001). It was
also shown that overexpression of Disp can increase the yield of Hh protein in
the culture medium (Ma et al.,
2002
). These experiments suggest that Hh can be directly secreted
from its expression cells. Can secreted Hh proteins freely diffuse to its
receiving cells through extracellular spaces? To address this issue, we have
conducted detailed analyses for Hh signalling in the complete absence of HS
GAG using sfl and ttv or absence of glypicans using
dally-dly. We show that a narrow strip (one cell diameter in width)
of sfl or ttv, or dally-dly mutant cells prevents
the transpassing of the Hh signal (Fig.
4). We also show that Hh staining disappears in sfl
mutant clones, except at a residual level in the posterior-most row of cells
(Fig. 6C). Based on these
observations, we favour a model in which Hh movement is regulated by a
cell-to-cell mechanism rather than by free diffusion.
Our results further suggest that Hh movement is independent of
dynamin-mediated endocytosis which has been shown to be involved in the
transportation of morphogen molecules
(Seto et al., 2002) such as
Dpp (Entchev et al., 2000
) and
Wg (Bejsovec and Wieschaus,
1995
; Moline et al.,
1999
). We found that a blockage of dynamin function did not
eliminate Hh movement and its subsequent signalling; instead, it led to a
striking reduction of punctate particles of Hh staining and an accumulation of
cell-surface Hh protein (Fig.
5). We also observed expanded Ptc expression domain when
dynamin-mediated endocytosis is blocked
(Fig. 5). These new findings
provide compelling evidence that dynamin-mediated endocytosis is not required
for Hh movement and its subsequent signalling, but is involved in Ptc-mediated
internalization of the Hh protein.
A model of Hh movement
Several mechanisms have been proposed to explain morphogen transport across
a field of cells. These mechanisms include (1) free diffusion, (2) active
transport by planar transcytosis, (3) cytonemes, (4) argosomes
(Entchev et al., 2000;
Ramirez-Weber and Kornberg,
1999
; Teleman et al.,
2001
; Vincent and Dubois,
2002
). Our results suggest that Hh moves through a cell-to-cell
mechanism rather than free diffusion. Furthermore, we demonstrate that
dynamin-mediated endocytosis is unlikely to be involved in Hh movement. On the
basis of our findings, we propose the following model by which the HSPGs Dally
and Dly may regulate the cell-to-cell movement of the Hh protein across a
field of cells. In this model (Fig.
7), Hh is released by Disp from its producing cells and is
immediately captured by the GAG chains of glypicans on the cell surface. The
differential concentration of Hh proteins on the surface of producing cells
and receiving cells drives the unidirectional displacement of Hh from one GAG
chain to another towards more distant receiving cells. Within the same cell,
the transport of Hh may be facilitated by the lateral movement of glypicans on
the cell membrane. On the receiving cells, glypicans may present Hh to Ptc,
which then mediates the internalization of Hh. Glypican mutant cells can not
relay Hh proteins further as they lack HS GAG on the surface. However, they
are able to respond to the Hh signal because Ptc may contact the Hh on the
membrane of the adjacent wild-type cells. Further studies are needed to
determine whether other mechanism(s) including cytonemes and argosomes are
also involved in Hh movement.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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Amanai, K. and Jiang, J. (2001). Distinct roles of Central missing and Dispatched in sending the Hedgehog signal. Development 128,5119 -5127.[Medline]
Azpiazu, N., Lawrence, P. A., Vincent, J. P. and Frasch, M. (1996). Segmentation and specification of the Drosophila mesoderm. Genes Dev. 10,3183 -3194.[Abstract]
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.
Bejsovec, A. and Wieschaus, E. (1995).
Signaling activities of the Drosophila wingless gene are separately mutable
and appear to be transduced at the cell surface.
Genetics 139,309
-320.
Belenkaya, T. Y., Han, C., Standley, H. J., Lin, X., Houston,
D., Heasman, J. and Lin, X. (2002). pygopus
encodes a nuclear protein essential for Wingless/Wnt signaling.
Development 129,4089
-4101.
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]
Bernfield, M., Gotte, M., Park, P. W., Reizes, O., Fitzgerald, M. L., Lincecum, J. and Zako, M. (1999). Functions of cell surface heparan sulfate proteoglycans. Annu. Rev. Biochem. 68,729 -777.[CrossRef][Medline]
Bhanot, P., Fish, M., Jemison, J. A., Nusse, R., Nathans, J. and
Cadigan, K. M. (1999). Frizzled and Dfrizzled-2
function as redundant receptors for Wingless during Drosophila embryonic
development. Development
126,4175
-4186.
Blair, S. S. (1992). Engrailed expression in the anterior lineage compartment of the developing wing blade of Drosophila. Development 115,21 -33.[Abstract]
Briscoe, J., Chen, Y., Jessell, T. M. and Struhl, G. (2001). A hedgehog-insensitive form of patched provides evidence for direct long-range morphogen activity of sonic hedgehog in the neural tube. Mol. Cell 7,1279 -1291.[CrossRef][Medline]
Burke, R., Nellen, D., Bellotto, M., Hafen, E., Senti, K. A., Dickson, B. J. and Basler, K. (1999). Dispatched, a novel sterol-sensing domain protein dedicated to the release of cholesterol-modified hedgehog from signaling cells. Cell 99,803 -815.[Medline]
Capdevila, J. and Guerrero, I. (1994). Targeted expression of the signaling molecule decapentaplegic induces pattern duplications and growth alterations in Drosophila wings. EMBO J. 13,4459 -4468.[Abstract]
Chamoun, Z., Mann, R. K., Nellen, D., von Kessler, D. P.,
Bellotto, M., Beachy, P. A. and Basler, K. (2001).
Skinny hedgehog, an acyltransferase required for palmitoylation and activity
of the hedgehog signal. Science
293,2080
-2084.
Chen, C. M. and Struhl, G. (1999). Wingless
transduction by the Frizzled and Frizzled2 proteins of Drosophila.
Development 126,5441
-5452.
Chen, M. S., Obar, R. A., Schroeder, C. C., Austin, T. W., Poodry, C. A., Wadsworth, S. C. and Vallee, R. B. (1991). Multiple forms of dynamin are encoded by shibire, a Drosophila gene involved in endocytosis. Nature 351,583 -586.[CrossRef][Medline]
Chen, Y. and Struhl, G. (1996). Dual roles for patched in sequestering and transducing Hedgehog. Cell 87,553 -563.[Medline]
Chou, T. B. and Perrimon, N. (1996). The
autosomal FLP-DFS technique for generating germline mosaics in Drosophila
melanogaster. Genetics
144,1673
-1679.
Duffy, J. B., Harrison, D. A. and Perrimon, N.
(1998). Identifying loci required for follicular patterning using
directed mosaics. Development
125,2263
-2271.
Entchev, E. V., Schwabedissen, A. and Gonzalez-Gaitan, M. (2000). Gradient formation of the TGF-beta homolog Dpp. Cell 103,981 -991.[Medline]
Forbes, A. J., Nakano, Y., Taylor, A. M. and Ingham, P. W. (1993). Genetic analysis of hedgehog signalling in the Drosophila embryo. Development Suppl.,115 -124.
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]
Gallet, A., Rodriguez, R., Ruel, L. and Therond, P. P. (2003). Cholesterol modification of hedgehog is required for trafficking and movement, revealing an asymmetric cellular response to hedgehog. Dev. Cell 4,191 -204.[Medline]
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]
Goodrich, L. V., Johnson, R. L., Milenkovic, L., McMahon, J. A. and Scott, M. P. (1996). Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by Hedgehog. Genes Dev. 10,301 -312.[Abstract]
Hatini, V. and DiNardo, S. (2001). Distinct signals generate repeating striped pattern in the embryonic parasegment. Mol. Cell 7,151 -160.[Medline]
Ingham, P. W. and McMahon, A. P. (2001).
Hedgehog signaling in animal development: paradigms and principles.
Genes Dev. 15,3059
-3087.
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.
Jeong, J. and McMahon, A. P. (2002).
Cholesterol modification of Hedgehog family proteins. J. Clin.
Invest. 110,591
-596.
Jiang, J. and Struhl, G. (1995). Protein kinase A and hedgehog signaling in Drosophila limb development. Cell 80,563 -572.[Medline]
Khare, N. and Baumgartner, S. (2000). Dally-like protein, a new Drosophila glypican with expression overlapping with wingless. Mech. Dev. 99,199 -202.[CrossRef][Medline]
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]
Lee, J. D. and Treisman, J. E. (2001). Sightless has homology to transmembrane acyltransferases and is required to generate active Hedgehog protein. Curr. Biol. 11,1147 -1152.[CrossRef][Medline]
Lee, J. J., Ekker, S. C., von Kessler, D. P., Porter, J. A., Sun, B. I. and Beachy, P. A. (1994). Autoproteolysis in hedgehog protein biogenesis. Science 266,1528 -1537.[Medline]
Lin, X., Buff, E. M., Perrimon, N. and Michelson, A. M.
(1999). Heparan sulfate proteoglycans are essential for FGF
receptor signaling during Drosophila embryonic development.
Development 126,3715
-3723.
Lin, X. and Perrimon, N. (1999). Dally cooperates with Drosophila Frizzled 2 to transduce Wingless signalling. Nature 400,281 -284.[CrossRef][Medline]
Lin, X. and Perrimon, N. (2000). Role of heparan sulfate proteoglycans in cell-cell signaling in Drosophila. Matrix Biol. 19,303 -307.[CrossRef][Medline]
Lin, X. and Perrimon, N. (2003). Developmental roles of heparan sulfate proteoglycans in Drosophila. Glycoconj. J. 19,363 -368.[CrossRef]
Lind, T., Tufaro, F., McCormick, C., Lindahl, U. and Lidholt,
K. (1998). The putative tumor suppressors EXT1 and EXT2 are
glycosyltransferases required for the biosynthesis of heparan sulfate.
J. Biol. Chem. 273,26265
-26268.
Lum, L., Yao, S., Mozer, B., Rovescalli, A., von Kessler, D.,
Nirenberg, M. and Beachy, P. A. (2003). Identification of
Hedgehog pathway components by RNAi in Drosophila cultured cells.
Science 299,2039
-2045.
Ma, Y., Erkner, A., Gong, R., Yao, S., Taipale, J., Basler, K. and Beachy, P. A. (2002). Hedgehog-mediated patterning of the mammalian embryo requires transporter-like function of dispatched. Cell 111,63 -75.[Medline]
Marigo, V., Scott, M. P., Johnson, R. L., Goodrich, L. V. and
Tabin, C. J. (1996). Conservation in hedgehog signaling:
induction of a chicken patched homolog by Sonic hedgehog in the developing
limb. Development 122,1225
-1233.
McCormick, C., Leduc, Y., Martindale, D., Mattison, K., Esford, L. E., Dyer, A. P. and Tufaro, F. (1998). The putative tumour suppressor EXT1 alters the expression of cell-surface heparan sulfate. Nat. Genet. 19,158 -161.[CrossRef][Medline]
Micchelli, C. A., The, I., Selva, E., Mogila, V. and Perrimon, N. (2002). Rasp, a putative transmembrane acyltransferase, is required for Hedgehog signaling. Development 129,843 -851.[Medline]
Moline, M. M., Southern, C. and Bejsovec, A.
(1999). Directionality of wingless protein transport influences
epidermal patterning in the Drosophila embryo.
Development 126,4375
-4384.
Motzny, C. K. and Holmgren, R. (1995). The Drosophila cubitus interruptus protein and its role in the wingless and hedgehog signal transduction pathways. Mech. Dev. 52,137 -150.[CrossRef][Medline]
Mullor, J. L., Calleja, M., Capdevila, J. and Guerrero, I.
(1997). Hedgehog activity, independent of decapentaplegic,
participates in wing disc patterning. Development
124,1227
-1237.
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.
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]
Park, Y., Rangel, C., Reynolds, M. M., Caldwell, M. C., Johns, M., Nayak, M., Welsh, C. J., McDermott, S. and Datt, S. (2003). Drosophila perlecan modulates FGF and hedgehog signals to activate neural stem cell division. Dev. Biol. 253,247 -257.[CrossRef][Medline]
Patel, N. H., Martin-Blanco, E., Coleman, K. G., Poole, S. J., Ellis, M. C., Kornberg, T. B. and Goodman, C. S. (1989). Expression of engrailed proteins in arthropods, annelids, and chordates. Cell 58,955 -968.[Medline]
Pepinsky, R. B., Zeng, C., Wen, D., Rayhorn, P., Baker, D. P.,
Williams, K. P., Bixler, S. A., Ambrose, C. M., Garber, E. A.,
Miatkowski, K. et al. (1998). Identification of a palmitic
acid-modified form of human Sonic hedgehog. J. Biol.
Chem. 273,14037
-14045.
Perrimon, N. and Bernfield, M. (2000). Specificities of heparan sulphate proteoglycans in developmental processes. Nature 404,725 -728.[CrossRef][Medline]
Porter, J. A., Ekker, S. C., Park, W. J., von Kessler, D. P., Young, K. E., Chen, C. H., Ma, Y., Woods, A. S., Cotter, R. J., Koonin, E. V. et al. (1996a). Hedgehog patterning activity: role of a lipophilic modification mediated by the carboxy-terminal autoprocessing domain. Cell 86,21 -34.[Medline]
Porter, J. A., Young, K. E. and Beachy, P. A.
(1996b). Cholesterol modification of hedgehog signaling proteins
in animal development. Science
274,255
-259.
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]
Rulifson, E. J., Wu, C. H. and Nusse, R. (2000). Pathway specificity by the bifunctional receptor frizzled is determined by affinity for wingless. Mol. Cell 6, 117-126.[Medline]
Seto, E. S., Bellen, H. J. and Lloyd, T. E.
(2002). When cell biology meets development: endocytic regulation
of signaling pathways. Genes Dev.
16,1314
-1336.
Siegfried, E. and Perrimon, N. (1994). Drosophila wingless: a paradigm for the function and mechanism of Wnt signaling. BioEssays 16,395 -404.[Medline]
Slusarski, D. C., Motzny, C. K. and Holmgren, R.
(1995). Mutations that alter the timing and pattern of cubitus
interruptus gene expression in Drosophila melanogaster.
Genetics 139,229
-240.
Spring, J., Paine-Saunders, S. E., Hynes, R. O. and Bernfield, M. (1994). Drosophila syndecan: conservation of a cell-surface heparan sulfate proteoglycan. Proc. Natl. Acad. Sci. USA 91,3334 -3338.[Abstract]
Strigini, M. and Cohen, S. M. (1997). A
Hedgehog activity gradient contributes to AP axial patterning of the
Drosophila wing. Development
124,4697
-4705.
Struhl, G., Barbash, D. A. and Lawrence, P. A.
(1997). Hedgehog acts by distinct gradient and signal relay
mechanisms to organise cell type and cell polarity in the Drosophila abdomen.
Development 124,2155
-2165.
Tabata, T. and Kornberg, T. B. (1994). Hedgehog is a signaling protein with a key role in patterning Drosophila imaginal discs. Cell 76,89 -102.[Medline]
Taylor, A. M., Nakano, Y., Mohler, J. and Ingham, P. W. (1993). Contrasting distributions of patched and hedgehog proteins in the Drosophila embryo. Mech. Dev. 42, 89-96.[CrossRef][Medline]
Teleman, A. A. and Cohen, S. M. (2000). Dpp gradient formation in the Drosophila wing imaginal disc. Cell 103,971 -980.[Medline]
Teleman, A. A., Strigini, M. and Cohen, S. M. (2001). Shaping morphogen gradients. Cell 105,559 -562.[CrossRef][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., Fox, B. and Selleck, S. B.
(2000). Structural analysis of glycosaminoglycans in animals
bearing mutations in sugarless, sulfateless, and tout-velu. Drosophila
homologues of vertebrate genes encoding glycosaminoglycan biosynthetic
enzymes. J. Biol. Chem.
275,21856
-21861.
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]
van der Bliek, A. M. and Meyerowitz, E. M. (1991). Dynamin-like protein encoded by the Drosophila shibire gene associated with vesicular traffic. Nature 351,411 -414.[CrossRef][Medline]
Vervoort, M., Crozatier, M., Valle, D. and Vincent, A. (1999). The COE transcription factor Collier is a mediator of short-range Hedgehog-induced patterning of the Drosophila wing. Curr. Biol. 9,632 -639.[CrossRef][Medline]
Vincent, J. P. and Dubois, L. (2002). Morphogen transport along epithelia, an integrated trafficking problem. Dev. Cell 3,615 -623.[Medline]
Voigt, A., Pflanz, R., Schafer, U. and Jackle, H. (2002). Perlecan participates in proliferation activation of quiescent Drosophila neuroblasts. Dev. Dyn. 224,403 -412.[CrossRef][Medline]
Xu, T. and Rubin, G. M. (1993). Analysis of
genetic mosaics in developing and adult Drosophila tissues.
Development 117,1223
-1237.
Zecca, M., Basler, K. and Struhl, G. (1995).
Sequential organizing activities of engrailed, hedgehog and decapentaplegic in
the Drosophila wing. Development
121,2265
-2278.
Zeng, X., Goetz, J. A., Suber, L. M., Scott, W. J., Jr, Schreiner, C. M. and Robbins, D. J. (2001). A freely diffusible form of Sonic hedgehog mediates long-range signalling. Nature 411,716 -720.[CrossRef][Medline]