1 Division of Developmental Biology, Cincinnati Children's Hospital Medical
Center, Cincinnati, OH 45229, USA
2 Graduate Program in Molecular and Developmental Biology, University of
Cincinnati College of Medicine, Cincinnati, OH 45229, USA
3 Graduate Program in Neuroscience, University of Cincinnati College of
Medicine, Cincinnati, OH 45229, USA
Author for correspondence (e-mail:
linyby{at}chmcc.org)
Accepted 15 December 2004
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SUMMARY |
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Key words: Heparan sulfate proteoglycans, Exostosin (EXT), tout-velu (ttv), botv of tout-velu (botv), sister of tout-velu (sotv), Wingless (Wg), Hedgehog (Hh), Decapentaplegic (Dpp), Drosophila
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Introduction |
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In the past few years, genetic studies in Drosophila have
demonstrated the crucial roles of heparan sulfate proteoglycans (HSPG) in
signalling events controlled by secreted Wg, Hh and Dpp morphogens
(Lander and Selleck, 2000;
Lin and Perrimon, 2000
;
Nybakken and Perrimon, 2002
;
Perrimon and Bernfield, 2000
).
HSPGs consist of a protein core to which heparan sulfate (HS)
glycosaminoglycan (GAG) chains are attached
(Bernfield et al., 1999
;
Esko and Selleck, 2002
;
Perrimon and Bernfield, 2000
).
The biosynthesis of HS GAG chains is initiated by the formation of a
GAG-protein linkage region consisting of a tetrasaccharide
(-GlcAß1-3Galß1-3Galß1-4Xylß-O-) attached to
specific serine residues in a proteoglycan core protein
(Bernfield et al., 1999
;
Esko and Selleck, 2002
).
Following the transfer of
-GlcNAc as the first N-acetylhexosamine unit
to this linkage region, HS co-polymerases add alternating ß1-4-linked
GlcA and
1-4-linked GlcNAc residues, generating HS GAG chains of 100 or
more sugar units in length (Esko and
Selleck, 2002
). Biochemical studies have demonstrated that both
the attachment of the first
-GlcNAc to the GAG-protein linkage region
and the subsequent polymer formation are catalyzed by members of the
hereditary multiple exostoses (EXT) gene family of tumor supressors
(Esko and Selleck, 2002
;
Zak et al., 2002
). In
vertebrates, the EXT gene family consists of EXT1, EXT2, and three
EXT-like genes designated EXTL1, EXTL2 and EXTL3
(Zak et al., 2002
). Human
mutations in EXT1 and EXT2 are associated with hereditary
multiple exostoses (HME), a benign bone tumor characterized by multiple
cartilage-capped outgrowths of various bones
(Ahn et al., 1995
;
Stickens et al., 1996
).
However, three EXT-like genes have not been demonstrated to be linked to
genetic disorder(s). A number of biochemical studies have shown that EXT1 and
EXT2 function as HS co-polymerases involved in HS polymerization
(Lind et al., 1998
;
McCormick et al., 2000
;
McCormick et al., 1998
;
Senay et al., 2000
;
Wei et al., 2000
). Recent
biochemical studies also demonstrated that both EXTL2 and
EXTL3 proteins possess enzymatic activities that can transfer
-GlcNAc to the GAG-protein linkage region and to intermediates of chain
polymerization, suggesting roles for these proteins in initiation and
polymerization reactions (Kim et al.,
2001
; Kitagawa et al.,
1999
; Zak et al.,
2002
). Despite intensive biochemical studies of the EXT family
proteins in the HS GAG biosynthesis, their relationship in HS GAG biosynthesis
and, in particular, their respective in vivo roles in development are largely
unknown.
The Drosophila genome contains three EXT family members. Previous
studies have shown that Tout-velu (Ttv), the Drosophila homologue of
mammalian EXT1, is required for Hh signalling
(Bellaiche et al., 1998;
The et al., 1999
). Hh movement
in the wing disc is defective in cells mutant for ttv. Further study
demonstrated that only cholesterol-modified Hh (Hh-Np), and not
cholesterol-unmodified (Hh-N), is dependent on Ttv function for its movement
(The et al., 1999
). Consistent
with this observation, a recent study showed that the movement of large
punctate structures containing Hh-Np across cells is contingent upon the
activity of Ttv (Gallet et al.,
2003
). The involvement of Ttv in HS GAG biosynthesis was also
demonstrated. HS GAG is strikingly reduced, but not completely eliminated, in
the ttv null embryo (The et al.,
1999
). Biochemical analysis further showed that HS GAG is markedly
reduced in ttv mutant larvae
(Toyoda et al., 2000
).
Together, these studies have demonstrated that Ttv is required for Hh movement
and involved in the HS GAG biosynthesis. Interestingly, it was shown that Ttv
is required specifically for Hh, but not for Wg and Fgf signalling
(The et al., 1999
), raising
the question of whether the other two Drosophila EXT members play
partially redundant roles with Ttv in signalling pathways other than Hh.
To understand the molecular mechanisms by which Wg, Hh and Dpp morphogen
gradients are regulated during wing development, we have conducted a genetic
screen for mutations associated with specific wing patterning defects
(Belenkaya et al., 2002). In
this paper, we report the identification and characterization of sister of
tout-velu (sotv; Ext2 FlyBase) and brother
of tout-velu (botv), encoding Drosophila homologues of
mammalian EXT2 and EXTL3, respectively. We show that Hh signalling and its
distribution are defective in either sotv or botv mutant
cells. We further demonstrate that all three Drosophila EXT proteins
(ttv, botv and sotv) are essential for Dpp signalling and
its morphogen distribution. Surprisingly, although all three
Drosophila EXT proteins are required for the proper extracellular Wg
distribution, Wg signalling is only defective in botv mutant or
ttv-sotv double mutant cells, but not in ttv nor
sotv mutant cells. We provide further biochemical evidence that Ttv
and Sotv form a complex and are co-localized in vivo. Our results provide new
insights into the functions of the EXT family proteins in morphogen signalling
during development.
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Materials and methods |
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Generation of marked clones for phenotypic analysis
Females with germline clones were generated using the autosomal `FLP-DFS'
technique (Chou and Perrimon,
1996) as described (Belenkaya
et al., 2002
; Hacker et al.,
1997
). Imaginal disc clones of mutant cells were generated as
described (Belenkaya et al.,
2002
; Hacker et al.,
1997
). To induce the expression of DsRed marker, third-instar
larvae were subsequently subjected to a second heat shock for 90 minutes at
37°C and allowed to recover for 5 hours at room temperature before
fixation and immunostaining. Below, we list the genotypes used in our
analyses.
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Molecular biology
The botv full-length cDNA was isolated by screening a 0-to-4 hour
Drosophila embryonic cDNA library
(Brown and Kafatos, 1988). The
coding region of botv cDNA with three haemagglutinin (HA) tags
in-frame at its C terminus was then cloned into the pUAST vector to generate
pUAST-botv-HA. The sotv cDNA was obtained from EST cDNA clone GH02288
(Invitrogen). The V5-tagged Sotv construct was generated by cloning the coding
region into the KpnI-EcoRV site of pAc5.1 V5-His C vector
(Invitrogen), in-frame with the V5 tag. To generate the pAc5.1-ttv-myc
construct, the ttv-myc fragment was amplified by PCR from the genomic
DNA of the UAS-ttv-myc line (The
et al., 1999
) and subcloned into the pAc5.1V5-His A vector.
HS-DsRed was generated by cloning the full-length
(BamHI-SpecI) DsRed T1 coding region from pDsRed expression
vector (Clontech) into BglII-XbaI sites of the pCasperR-hs
vector.
Immunoprecipitation and western blotting
Drosophila Schneider's S2 cells (1x107) were
transfected with 10 µg of corresponding expression vectors by the calcium
phosphate precipitation method. For the induction of Botv-HA cloned in pUAST
vector, 10 µg pUAST-botv-HA and 10 µg of pArmadillo-Gal4
(Klueg et al., 2002) was
co-transfected. Cells were harvested 60 hours later, and lyzed in 1.5 ml of 20
mM Tris-HCl (pH 7.4), 2% Triton X-100, 150 mM NaCl and 7.5 ml proteinase
inhibitor tablet (Roche Molecular Biochemicals) on ice for 20 minutes. After
clearance, one half of each lysate was used for immunoprecipitation with 1
µg antibodies for 3 hours at 4°C and then incubated for an additional
1.5 hours in the presence of 12.5 µl bed volume of protein G sepharose
(Amersham Pharmacia). Immunoprecipitates were washed three times with 10 mM
Tris-HCl (pH 7.4), 0.2% Triton X-100, 150 mM NaCl, 2 mM EDTA and 1 µl
proteinase inhibitor (Sigma), and twice with 10 mM Tris-HCl (pH 7.4). Western
blotting was carried out as described
(Belenkaya et al., 2002
).
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Results |
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botv and sotv encode the Drosophila homologues of mammalian EXTL3 and EXT2, respectively, and are required for HS GAG biosynthesis
botv and sotv were subsequently mapped to the cytological
positions 56A-56C and 52F, respectively (see Materials and methods). Searches
of annotated Drosophila genome databases identified a
Drosophila EXT-like gene (CG15110) in 56A-56C and a
Drosophila EXT2 (DEXT2; CG8433) in 52F. The
Drosophila genome contains three EXT genes including ttv,
CG15110 and CG8433. Based on the similarities of botv and
sotv with ttv in both wing and embryonic cuticle defects, we
suspected that the CG15110 and CG8433 transcripts may encode Botv and Sotv,
respectively. Two lines of evidence strongly suggest that this is indeed the
case. First, we used the RNA interference (RNAi) method
(Kennerdell and Carthew, 1998)
to perturb CG15110 and CG8433 transcripts. Embryos injected with either
CG15110 or CG8433 double-stranded RNA showed segment-polarity defects (data
not shown). Second, all the sequenced alleles of botv and
sotv have mutations in the CG15110 and CG8433 genes, respectively
(selected alleles are shown in Fig.
2A).
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Biochemical studies have demonstrated that EXT family proteins are required
for HS GAG biosynthesis (Esko and Selleck,
2002; Zak et al.,
2002
). Biosynthesis of HS GAG chains is strikingly reduced in
ttv mutant embryos (The et al.,
1999
) and larvae (Toyoda et
al., 2000
). We analyzed levels of HS GAG chains in clones mutant
for ttv, botv and sotv in the wing disc. In wild-type cells,
HS GAG staining was found in punctate particles as well as on the membrane
(Fig. 3). Consistent with
previous studies in embryos, HS GAG staining in ttv mutant clones was
strikingly reduced (Fig.
3A,A'), suggesting that Ttv is required for HS GAG
biosynthesis in the wing disc as well. We also observed similar reductions in
HS GAG staining in cells mutant for sotv
(Fig. 3B,B') or
botv (Fig.
3C,C'). On the basis of these observations, we conclude that
all three Drosophila EXT proteins are indispensable for the
biosynthesis of HS GAG chains.
Botv and Sotv are required for Hh signalling in the wing disc
Previous studies have demonstrated the essential role of Ttv in Hh
signalling in the wing disc (Bellaiche et
al., 1998). Hh movement is blocked in A compartment cells mutant
for ttv (Bellaiche et al.,
1998
). Two lines of evidence support the idea that Botv and Sotv
are also required for Hh signalling. First, the level of Cubitus interruptus
(Ci) is stabilized by Hh signalling in about 8-10 cells in the AP border
(Aza-Blanc et al., 1997
;
Methot and Basler, 1999
;
Motzny and Holmgren, 1995
).
However, Ci stabilization is strikingly reduced in anterior cells mutant for
sotv (Fig.
4B,B') or botv
(Fig. 4C,C'). Second,
whereas Hh proteins are present as punctate particles in anterior cells at the
AP border, these punctate particles are absent within sotv
(Fig. 4E,E') and
botv clones (Fig.
4F,F'), except in the first row of cells facing the P
compartment. These results suggest that similar to Ttv, Sotv and Botv
activities are required for Hh movement in its receiving cells. In the absence
of either Sotv or Botv, Hh can only move into the first row of cells
immediately adjacent to the Hh-expressing cells, and fails to move
further.
Ttv, Sotv and Botv are required for Dpp signalling and its gradient distribution
In the wing disc, Dpp functions as a long-range morphogen to control the
growth and patterning of cells in the AP axis
(Lecuit et al., 1996;
Nellen et al., 1996
). Dpp
signalling is shown to activate its downstream signalling component Mad in a
concentration-dependent manner (Tanimoto
et al., 2000
). Recently, the Dpp morphogen gradient has been
visualized directly using GFP-Dpp fusion proteins that retain signalling
activity (Entchev et al.,
2000
; Teleman and Cohen,
2000
).
Vein deletions are the most striking defects associated with wing-bearing
clones mutant for ttv, sotv and botv
(Fig. 1B,C,D). As Dpp
signalling is required for vein formation
(de Celis et al., 1996;
Ray and Wharton, 2001
), we
investigated whether Dpp signalling and its gradient distribution were
defective in clones mutant for ttv, sotv and botv. We first
examined Dpp signalling activity by visualizing the activated form of Mad
(p-Mad), which is phosphorylated by the activated Dpp receptor Thickveins
(Tkv) in response to Dpp signalling
(Tanimoto et al., 2000
). In
the wild-type wing disc (Fig.
5A), p-Mad levels were high in the central region of the wing disc
and gradually decline towards the A and P distal cells. p-Mad levels were
lower at the AP boundary owing to the reduced expression of tkv
(Tanimoto et al., 2000
). p-Mad
levels were strikingly reduced in either A or P cells mutant for ttv,
sotv or botv (Fig.
4B-D'), providing evidence that all three
Drosophila EXT proteins are required for Dpp signalling. We further
tested whether the EXT proteins control Dpp signalling by regulating Dpp
morphogen distribution. For this purpose, we expressed GFP-Dpp in the
endogenous dpp expression domain using DppGAL4
and analyzed GFP-Dpp distribution in clones mutant for ttv, sotv or
botv. In the wild-type background, GFP-Dpp exhibits a gradient
pattern in both the A and P compartment
(Fig. 5E). However, levels of
GFP-Dpp are reduced in clones mutant for ttv, sotv or botv
(Fig. 5F-H'). Together,
these results argue that Ttv, Sotv and Botv promote Dpp signalling by
modulating its morphogen distribution.
Distinct roles of Botv from those of Ttv and Sotv in Wg signalling in the wing disc
In the wing disc, Wg forms a long-range gradient and acts both at short and
long range to regulate the expression of several target genes in different
spatial domains (Neumann and Cohen,
1997; Strigini and Cohen,
2000
; Zecca et al.,
1996
). The homeodomain protein Distal-less (Dll)
(Neumann and Cohen, 1997
;
Zecca et al., 1996
) and the
zinc-finger protein Senseless (Sens) (Nolo
et al., 2000
; Parker et al.,
2002
) are the long- and short-range targets of the Wg morphogen,
respectively (Fig.
6A,A'). We have previously shown that HSPGs are required for
Wg short- and long-range signalling, as well as for its extracellular
distribution in the wing disc (Baeg et al.,
2001
; Lin and Perrimon,
1999
). However, a previous study on Ttv demonstrated that Ttv is
not involved in Wg signalling during embryogenesis and in the wing disc,
suggesting that Ttv selectively participates in morphogen signalling
(The et al., 1999
). To
evaluate the specificity of the three Drosophila EXT genes in
morphogen signalling in the wing disc, we examined Wg short- and long-range
signalling activities, as well as its morphogen distribution in clones mutant
for ttv, sotv and botv.
We observed reductions in the ranges of Dll expression in ttv, sotv and botv mutant cells (Fig. 6B,C,D). These defects were fully penetrant, suggesting that all three EXT proteins are normally required for Wg long-range activity. Interestingly, we found that Dll levels were not reduced in regions close to the DV boundary within the clones of ttv and sotv mutant cells; however, they were significantly reduced in the same region within the botv mutant clone (Fig. 6B,C,D). These results suggest that both the range of Wg action and its signalling are affected in the botv mutant clone; however, only the range of Wg action, and not its signalling per se, is reduced in the ttv or sotv clones. Consistent with this, we found that Sens expression was diminished in botv clones, but not in ttv nor sotv clones, confirming that Wg signalling is defective only in the botv mutant, and not in ttv or sotv mutant (Fig. 6B',C',D'). The observed defects in the ranges of Dll expression in ttv, sotv and botv mutant clones could be due to reduced levels of Wg morphogen. We tested this by staining extracellular Wg. The result was in agreement with the Dll data (Fig. 6F-H'). On one hand, the range of extracellular Wg distribution was reduced in clones mutant for ttv, sotv and botv. On the other hand, extracellular Wg was maintained at some levels in the regions close to DV boundary in the ttv or sotv clone, however, it was absent in the same regions within the botv clone, except at the surface of Wg-expressing cells. Together, these results suggest that all three Drosophila EXT proteins are required for the long-range distribution of Wg protein. However, Wg signalling is only defective in the botv mutant, and not in ttv or sotv mutant cells.
One apparent explanation for this is that Ttv and Sotv are functionally redundant in Wg signalling. To test this, we examined Sens expression in clones of ttv-sotv double mutants and found that, indeed, Sens expression was diminished (Fig. I-J'). A virtually identical result in Sens expression was observed in ttv-sotv-botv triple mutant cells (data not shown). Taken together, our findings suggest that all three EXT proteins are required for proper extracellular Wg distribution. However, whereas Botv is independently required for Wg signalling, Ttv and Sotv are redundant in Wg signalling.
Biochemical interactions and subcellular localization of the Drosophila EXT proteins
The distinct roles of Botv from those of Ttv and Sotv in Wg signalling
suggest that they may have unique functions in the biosynthesis of HS GAG
chains. In vertebrates, direct enzymatic assay for vertebrate EXTL3
demonstrates that its GlcNAc transferase activities are involved in both the
initiation and polymerization reactions
(Kim et al., 2001), whereas
several biochemical studies suggested that EXT1 and EXT2 may function as a HS
GAG co-polymerase in which both EXT1 and EXT2 serve as subunits essential for
the activity (McCormick et al.,
2000
; McCormick et al.,
1998
; Senay et al.,
2000
; Wei et al.,
2000
; Zak et al.,
2002
). As Ttv and Sotv are most similar to the vertebrate EXT1 and
EXT2, respectively, we anticipated that Ttv and Sotv may function as a HS GAG
co-polymerase whose full activity requires both Ttv and Sotv. By contrast,
Botv, a homologue of vertebrate EXTL3, may participate in the initiation step
of HS GAG biosynthesis, which is distinct from the role of Ttv and Sotv.
To determine whether Ttv and Sotv function as subunits for the HS copolymerase, we performed a co-immunoprecipitation experiment to examine whether Ttv and Sotv form a complex in cells. Myc-tagged Ttv and V5-tagged Sotv were expressed either individually or in combination in Drosophila S2 cells. Upon immunoprecipitation of Myc-tagged Ttv from the cellular lysate of transfected cells, the Sotv protein could be detected by western blotting in the immunoprecipitate (Fig. 7A). Interestingly, we did not observe an interaction between Ttv and Sotv when cellular lysates from individually transfected cells were mixed and immunoprecipitated (Fig. 7A, lane labeled m), indicating that Ttv and Sotv cannot associate ex vivo. We further conducted similar experiments to examine the association of Botv with Ttv or Sotv (Fig. 7B,C). In these cases, no interactions were detected. These data suggest that Ttv and Sotv are physically associated in vivo, but that they do not form complexes with Botv.
|
We further tested whether overexpression of Ttv can replace the function of Sotv and Botv. For this purpose, we ectopically expressed Ttv in the hairy expression domain by using hairyGal4 IJ3 in either sotv or botv null mutant embryos. Ectopic expression of Myc-tagged Ttv fully rescued the cuticle patterning of the ttv mutant embryo in the hairy domain (Fig. 7H), but was not able to rescue cuticle defects associated with sotv or botv null mutant embryos (Fig. 7I,J). This result further indicates that individual EXT members play indispensable roles in HS GAG biosynthesis.
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Discussion |
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Roles of the Drosophila EXT family proteins in Hh and Dpp morphogen signalling
Previous studies have demonstrated that Ttv is involved in Hh movement
(Bellaiche et al., 1998;
Gallet et al., 2003
;
The et al., 1999
). Our results
in this work suggest that like Ttv, both Sotv and Botv are also required for
Hh movement. Interestingly, we found that Hh is detectable in the first row of
mutant cells immediately adjacent to its posterior-producing cells. We propose
that specific HSPGs modified by EXT family proteins are required for the
movement of Hh from its expressing cells into the anterior-receiving cells. In
the absence of EXT activities, Hh can be carried into the first row of mutant
cells, but fails to move further. Our results are consistent with previous
work that demonstrated that the first row of ttv mutant cells can
still transduce Hh signalling and can activate the expression of its
downstream target gene patched (ptc)
(Bellaiche et al., 1998
). It is
important to note that, while HSPGs modified by EXT family members are likely
to be required for the movement of Hh, they may also be involved in preventing
Hh from being degraded on the cell surface. In the absence of EXT proteins, Hh
may be degraded and therefore it cannot reach the wild-type cells anterior to
the clones of EXT mutant cells. It remains to be determined whether both
mechanisms are involved in Hh transport.
We provide strong evidence for the involvement of the three EXT proteins in
Dpp signalling and its morphogen distribution. Our results suggest that
specific HSPG(s) modified by the three EXT proteins may promote Dpp morphogen
signalling by modulating levels of Dpp morphogen ligands in Dpp-receiving
cells. Previous studies have implicated a role for Dally, a
Drosophila glypican member of HSPGs, in Dpp signalling in the
development of imaginal discs (Fujise et
al., 2003; Jackson et al.,
1997
). Elevated expression of Dally in the wing disc can promote
Dpp signalling, as assayed by p-Mad levels
(Fujise et al., 2003
). It was
proposed that Dally may act as a co-receptor for Dpp in activating its
signalling. Our results in this work suggest that HS GAG chains control Dpp
signalling by modulating levels of Dpp morphogen in its receiving cells. We
propose that the three EXT proteins are required for the biosynthesis of HS
GAG chains on the Dally protein and that at least one of the roles of Dally in
Dpp signalling is to modulate levels of the Dpp morphogen on its receiving
cells.
Role of HSPGs in Wg morphogen distribution and its signalling
Perhaps one of the most interesting findings in this work is the
differential roles of the three EXT proteins in Wg morphogen signalling.
Although mutations in any of the three EXT genes lead to reduced ranges of
extracellular Wg distribution, we found that Wg signalling was defective only
in botv mutant cells, and not in either ttv or sotv
mutant cells. However, ttv-sotv double mutants showed virtually
identical defects in Wg signalling to those of botv mutants. This
qualitative difference between Botv and Ttv or Sotv in Wg signalling is
unexpected as mutations in all three genes led to striking reductions in Hh
and Dpp signalling.
Previous studies have implicated HSPGs in both Wg signalling and its
morphogen gradient distribution. Embryos null for either sugarless or
sulfateless, exhibit defects in Wg signalling in various tissues
(Binari et al., 1997;
Hacker et al., 1997
;
Haerry et al., 1997
;
Lin and Perrimon, 1999
). The
Drosophila glypican Dally has been shown to be required for Wg
signalling (Fujise et al.,
2001
; Lin and Perrimon,
1999
; Tsuda et al.,
1999
). A reduction of dally activity can block the Wg
signalling activity induced by overexpression of the Wg receptor
Drosophila frizzled 2 (fz2) in the wing disc
(Lin and Perrimon, 1999
).
Interestingly, recent studies have demonstrated that Notum (Wingful), a
putative Drosophila pectin acetylesterase, can inhibit Wg signalling
activity by modulating the activity of the heparin sulfate proteoglycans
Dally-like (Dly) and Dally (Gerlitz and
Basler, 2002
; Giraldez et al.,
2002
). Overexpression of Notum can block the signalling
activity of the Wg protein tethered to the cell surface by a transmembrane
domain from Neurotactin, suggesting that Notum inhibits the function of
proteoglycans that are involved in Wg signalling
(Gerlitz and Basler, 2002
).
The function of HSPGs in Wg morphogen gradient distribution has also been
demonstrated. In the wing disc, cells mutant for sulfateless showed a
reduction in the levels of extracellular Wg
(Baeg et al., 2001
). It was
shown that ectopic expression of Dly leads to the accumulation of
extracellular Wg protein (Baeg et al.,
2001
). Furthermore, loss of Notum function leads to increased
Wingless activity by altering the shape of the Wingless protein gradient
(Giraldez et al., 2002
).
Taken together, our results, along with previous studies, suggest that
HSPGs are involved in both Wg signalling reception and in extracellular Wg
morphogen distribution in the wing disc. We suggest that HSPGs have at least
two distinct functions in the wing disc: (1) in the distribution of the
extracellular Wg protein; and (2) as a co-receptor for Wg signalling. In this
regard, Botv is required both for Wg signalling and for its morphogen gradient
formation, whereas Ttv and Sotv are only required for the distribution of
extracellular Wg protein; they are functionally redundant in Wg signalling.
Consistent with our observations, a previous report on ttv showed
that, during embryogenesis, Wg signalling in the stomatogastric nervous system
(SNS) is not defective in ttv mutant embryos
(The et al., 1999). It remains
to be determined whether botv mutants and sotv-ttv double
mutants show effected Wg signalling in the SNS and in other tissues as
well.
Mechanisms and specificities of Ttv, Sotv and Botv in cell signalling
Previous analysis of Ttv has demonstrated its specificity in cell
signalling (The et al., 1999).
Although Hh signalling is defective in the ttv null mutant, neither
Wg nor Fgf signalling is altered (The et
al., 1999
). It was proposed that Ttv is required for the synthesis
of an Hh-specific HSPG. Consistent with the previous report, our results
demonstrated that Wg signalling is only defective in the ttv-sotv
double mutant, and is not altered in either ttv or sotv
single mutants. However, we also observed striking defects in both Dpp
signalling and the range of extracllular Wg protein distribution in
ttv and sotv mutants. Therefore, the previous view that Ttv
is involved only in Hh signalling should be revised.
To understand the molecular mechanisms by which the Drosophila EXT
proteins play distinct and collaborative roles in cell signalling, we
performed biochemical experiments to analyze their interactions and
subcellular localization. We found that Ttv and Sotv physically associate with
each other to form a complex, and that they have virtually identical
subcellular localizations. However, neither Ttv nor Sotv physically interact
with Botv. Botv also has a more diffusive staining in cells than Ttv and Sotv.
Consistent with our results, biochemical studies in vertebrates showed that
vertebrate EXT1 and EXT2 also physically associate with each other to form a
complex. Biochemical studies have further demonstrated that both EXT1 and EXT2
have GlcNAc and GlcA transferase activities when expressed independently,
although EXT1 has a more robust activity than does EXT2
(Lind et al., 1998;
McCormick et al., 2000
;
McCormick et al., 1998
;
Senay et al., 2000
;
Wei et al., 2000
). However,
co-expression of EXT1 and EXT2 has a synergistic effect on enzyme activities
(McCormick et al., 2000
;
Senay et al., 2000
). These
results led to a proposal that the fully active HS co-polymerase may be a
complex containing EXT1 and EXT2, in which both subunits are essential for the
activity (Zak et al., 2002
).
The functions of EXT-like proteins have also been investigated. EXTL3 appears
to be a bifunctional
GlcNAc transferase that can transfer GlcNAc to
both the linkage region and to intermediates during chain polymerization,
suggesting that EXTL3 is involved in both the initiation and polymerization of
HS GAG chains (Kim et al.,
2001
). Importantly, a recent biochemical study demonstrated that
Botv has
GlcNAc transferase activities that can transfer GlcNAc to both
the linkage region and to intermediates in chain polymerization
(Kim et al., 2002
).
On the basis of our genetic evidence and previous biochemical studies, we
propose that Ttv and Sotv are likely to function as co-polymerases required
for HS GAG polymerization, whereas Botv is likely to be involved in the
initiation of HS GAG and is possibly involved in HS GAG polymerization as
well. In the absence of Botv, no HS GAG chains are initiated and therefore
mutations in Botv disrupt all the functions of HS GAG chains. However, in the
absence of either Ttv or Sotv, initiation of HS GAG biosynthesis occurs, and
the residual activity of HS GAG polymerase(s), carried out by another member
(either Sotv or Ttv) together with Botv, may synthesize relatively short HS
GAG chains that could act as co-receptors for Wg signalling, but have less
capacity for maintaining the levels of secreted Wg, Hh and Dpp morphogen
proteins. When the activities of both Ttv and Sotv are removed in the
ttv-sotv double mutant, HS GAG polymerization may not occur because
of the lack of GlcA transferase activity, even though Botv is present. In
support of this view, previous biochemical studies demonstrated that short HS
GAG oligosaccharides have the capacity to form Fgf-Fgfr-HS complexes and to
stimulate Fgf signalling (Pellegrini,
2001). In this regard, in the absence of Ttv, both Wg and Fgf
signalling may occur. Consistent with this view, a previous study demonstrated
that Fgf signalling is not affected in ttv mutant embryos
(The et al., 1999
).
Biochemical studies on the activities of Ttv and Sotv as HS co-polymerases
will further validate this view. An alternative model is that fewer intact HS
GAG chains are synthesized in the absence of either Ttv or Sotv. Although this
is less likely to be the case, our current results can not exclude this
possibility.
EXT proteins and HME disease
Human mutations in EXT1 and EXT2 are associated with
hereditary multiple exostoses (HME), which is an autosomal dominant disorder
characterized by the formation of multiple cartilage-capped tumors (exostoses)
of various bones. Our results demonstrate the essential functions of both Ttv
(EXT1) and Sotv (EXT2) in regulating the activities of the three secreted
morphogen molecules Hh, Wg and Dpp. Wg and Dpp are the homologues of human WNT
and bone morphogen protein (BMP) molecules, respectively. As both WNT and BMP
family proteins have been shown to be essential for bone growth and
differentiation, our results suggest possible roles for WNT and BMP signalling
in the generation of HME diseases associated with EXT1 and
EXT2. Our new findings together with previous work on the role of Ttv
in Hh movement may provide new insights into the molecular mechanism(s)
associated with HME disease.
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ACKNOWLEDGMENTS |
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Footnotes |
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