Institute of Molecular and Cellular Biosciences, University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-0032, Japan
* Author for correspondence (e-mail: ttabata{at}ims.u-tokyo.ac.jp)
Accepted 30 September 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: EXT genes, tout-velu, sister of ttv, brother of ttv, Morphogen, Gradient formation, Hedgehog, Decapentaplegic, Wingless, Drosophila
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Although the influence of Dpp and Wg extends to the edges of the wing disc
(Capdevila and Guerrero, 1994;
Neumann and Cohen, 1997b
;
Zecca et al., 1995
) and Hh can
signal across long distances as well (Chen
and Struhl, 1996
), these proteins share an unexpected
characteristic; they are not readily soluble. The active form of Hh has
cholesterol covalently bound at its C-terminus
(Porter et al., 1996
) and an N
terminus that is palmitoylated (Pepinsky
et al., 1998
). Both modifications will probably anchor these
proteins in the membranes of the cells in which they are produced. Both Wg and
Dpp homolog TGF-ß bind to matrix proteins
(Reichsman et al., 1996
;
Taipale and Keski-Oja, 1997
).
In addition, Wg can also undergo lipid modification
(Willert et al., 2003
). Simple
diffusion thus appears inadequate to distribute these proteins over even short
distances.
Recently, several reports suggest that heparan sulfate proteoglycans
(HSPGs) play a key role in morphogen transport and/or signaling (reviewed by
Perrimon and Bernfield, 2000).
HSPGs are abundant cell surface molecules and are part of the extracellular
matrix. HSPGs consist of a protein core (such as syndecans and glypicans) to
which heparan sulfate glycosaminoglycan (HS GAG) chains are attached. GAG
chains are long unbranched polymers consisting of many sulfated disaccharides.
(A GAG synthesis diagram is shown in Fig.
1G).
|
Among the three GAG-biosynthetic genes, the ttv mutant is unique
in its selective effects on Hh signaling. Wg-directed events were unaffected
in ttv embryos and larvae
(Bellaiche et al., 1998;
The et al., 1999
). A possible
explanation accounting for this apparent selectivity is that HS GAGs
synthesized by other EXT genes may be sufficient to allow Wg pathways to
function (`quantitative model'). Alternatively, Hh-specific HSPGs may exist
and ttv might be required for their synthesis, whereas other EXT
genes might synthesize HSPGs specific for Wg signaling (`qualitative
model').
Here we describe novel mutants of sister of tout-velu (sotv) and brother of tout-velu (botv), as well as new alleles of ttv. sotv and botv, like ttv, encode EXT family proteins. In the clones of mutant cells, HSPGs biosynthesis was severely affected, and the morphogen signaling activities of Hh, Dpp and Wg were impaired to various degrees. Morphogen molecules were rarely seen in mutant clones, and had been accumulated at the wild-type cells that reside next to a mutant clone. These results suggest that HSPGs are required for proper transport of morphogens.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Mapping
One of three complementation groups that had similar phenotypes, failed to
complement the lethality of ttv, which has been reported to be a
Drosophila EXT family gene
(Bellaiche et al., 1998) and
two other groups failed to complement Df(2R)Jp8 and Df(2R)P34 which delete
52F5-9; 53A1 and 55E6-F3; 56C1, respectively. In and around these regions
there are two other Drosophila EXT genes, DEXT2
(Han et al., 2002
;
The et al., 1999
) and
DEXT3 (Han et al.,
2002
; Han et al.,
2001
; Kim et al.,
2002
). We sequenced all predicted coding regions of their genomic
DNA and identified SNPs in DEXT2 and DEXT3.
Clonal analysis
Clones of mutant cells were induced by Flippase-mediated mitotic
recombination (Xu and Rubin,
1993). First instar larvae were heat-shocked at 37°C for 1
hour and dissected 2-4 days after the heat-shock. Mutant clones were
identified by the loss of GFP expression. Genotypes of the flies used for
making mitotic clones were as follows: y w FLP122/+;
FRT42D y+ sha EXT
gene(s)/FRT42D y+ Ub-GFP or
wglacZ FRT42D Ub-GFP or dpp-lacZ
FRT42D Ub-GFP. For monitoring the Dpp-GFP distribution in the EXT
mutant clone, we dissected wing discs of y FLP122/+;
FRT42D ttv524
botv510/FRT42D hs-CD2;
UAS-dpp-GFP/dpp-GAL4 larvae.
Histochemistry
Imaginal disc staining was performed as described previously
(Tanimoto et al., 2000),
except for 3G10 staining: treatment of imaginal discs to expose the epitope
recognized by the 3G10 monoclonal antibody (Seikagaku) was done by incubating
fixed imaginal discs with 20 mU heparitinase I (Seikagaku) per 1 ml 100 mM
NaOAc, 3.3 mM CaCl2 for 1 hour at 37°C. After washing three
times with PBT, the samples were stained with 3G10 antibody, 1:100 in 2%
BSA/PBT.
The primary antibodies used were: rabbit anti-ß-gal antibody, 1:2000
(Cappel); mouse anti-CD2 antibody, 1:1000 (Cederlane); rat anti-Ptc antibody,
1:150 (gift from R. Johnson); rat anti-Sal antibody, 1:250 (gift from R.
Barrio); rabbit anti-p-Mad antibody PS1, 1:20,000
(Persson et al., 1998); rabbit
anti-Dll antibody, 1:300 (Panganiban et
al., 1995
); mouse anti-Wg antibody 4D4, 1:5000 (Developmental
Studies Hybridoma Bank); rabbit anti-Hh antibody NHhI, 1:1000 (raised against
the N-terminal amino acids 89-306 of Hh); rabbit anti-Dpp antibody, 1:5000
(gift from M. Hoffmann).
Secondary antibodies used were anti-mouse Cy3, anti-rabbit Cy3, anti-Rat Cy3, anti-rabbit Cy5 and anti-rat Cy5 (Jackson).
Immunofluorescent images were observed on a Zeiss confocal laser microscope 510.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Crosses to deficiency strains mapped the other two loci to the interval
52F5-9; 53A1 and 55E6-F3; 56C1, respectively. Near these regions, two
EXT-related genes have been identified by homology search: DEXT2,
sotv (CG8433, 52E11) (Han et al.,
2002; The et al.,
1999
) and DEXT3, botv (CG15110, 56B5)
(Han et al., 2002
;
Han et al., 2001
;
Kim et al., 2002
). However,
evidence for the function of these genes in an intact organism has been
lacking because no mutant has been identified. We sequenced all predicted
coding regions of these genes and identified deletions and SNPs in exons of
both DEXT2 and DEXT3
(Fig. 1F).
The predicted ORF of sotv encodes a protein with 717 amino acids.
Our Drosophila sotv326 mutant has a nonsense mutation at
glutamic acid 626 that cuts off 92 C-terminal residues
(Fig. 1F). This candidate gene
must represent sotv, because a transgene harboring a full-length
sotv cDNA driven by the ubiquitous promoter of the tubulin
alpha1 gene was found to completely rescue the lethality of
sotv326/Df(2R)Jp8 and other phenotypes associated with clones of sotv
mutation (data not shown). The Sotv sequence is similar to human EXT2 (44.8%
amino acid similarity), which also has GlcNAcT-II and GlcAT-II activities
(Senay et al., 2000).
The predicted botv encodes a protein with 972 residues and shares
45.8% sequence identity with human EXTL3. Three mutants of our third
complementation group, botv423,
botv510 and botv514 have nonsense
mutation at Leu 259, missense mutation at Pro 669 Leu, and Gly 821 Asp,
respectively (Fig. 1F). Human
EXTL3 has GlcNAcT-I and -II activities and is required for initiation of HS
biosynthesis. Previous studies have shown that a truncated soluble form of
Drosophila Botv protein has GlcNAcT-I and GlcNAcT-II activities when
expressed in COS-1 cells (Kim et al.,
2002).
Like most EXT family members, including Ttv, Sotv and Botv have three apparent domains: an N-terminal transmembrane domain; a DXD motif which is characteristic of glycosyltransferases and is required for binding divalent cations; and a large globular domain in the C-terminal region that will probably have enzymatic activity (Fig. 1F). Amino acid sequences of these Drosophila EXTs are related, with 26-32% amino acid overall similarity, and especially high homology in their C-terminal regions (Fig. 1H).
Animals transheterozygous for each EXT mutant (ttv524/ttv205, botv423/botv510, sotv326/Df(2R)Jp8) die as pupae with small eyes and legs (data not shown). In all cases, third instar larvae have smaller discs than the wild type, and in particular, their wing discs were narrower in anterior-posterior direction (data not shown). In situ hybridization revealed that third instar larvae express sotv and botv ubiquitously in wing discs as well as ttv (data not shown).
HSPG biosynthesis is defective in EXT mutants
To test whether sotv and botv function in HSPG
biosynthesis in flies, we stained wing imaginal discs with antibody 3G10 that
recognizes an epitope produced by heparitinase I digestion of HSPGs
(David et al., 1992). In the
wild-type wing discs, uniform staining was detected
(Fig. 2A). However in discs
with mutant clones, 3G10 staining was severely reduced in cells mutant for any
of the EXT genes (Fig. 2B-D).
These results indicate that HSPG biosynthesis in Drosophila requires
each of these genes.
|
Hh, Dpp and Wg signaling is defective in EXT mutants
Hh signaling
In the wing imaginal disc, Hh is only expressed in the posterior
compartment cells but diffuses into the anterior compartment
(Basler and Struhl, 1994;
Chen and Struhl, 1996
;
Lee et al., 1992
;
Tabata and Kornberg, 1994
)
where it upregulates expression of dpp and patched
(ptc). dpp responds to low Hh activity and is observed as
far as 15 cells away from the A/P boundary; ptc responds to higher
Hh-signaling activity and can be detected only up to five cells away
(Fig. 3A). We used dpp
and Ptc expression as reporters of the Hh signaling in EXT mutants.
|
Almost no change in level of dpp expression was detected when mutant clones grew along the boundary in the posterior compartment (arrow in Fig. 3D,E). This result suggests that neither sotv nor botv is required for Hh secretion, but rather that their functions are necessary for efficient movement of Hh. However on the same occasion, Ptc protein level was sometimes slightly decreased in the anterior cells along the A/P boundary (Fig. 3D,E). It is probably because ptc expression requires higher Hh signal than dpp expression, and is more sensitive to reduction in Hh protein level in posterior compartment, as described below (Fig. 6A). The same results were observed in ttv524 mutant clones (data not shown).
|
|
Wg signaling
A functional connection between Wg and HSPGs has been suggested by the
observed strong association of Wg proteins and sulfated proteoglycans in
tissue culture cells (reviewed by Lin and
Perrimon, 2000; Selleck,
2001
). In addition, Wg signaling is affected by defective HSPG
biosynthesis in sgl and sfl mutants, and in dally
and dlp mutants that alter HSPG core proteins. Wg is normally
expressed at the wing margin and controls patterning along the dorsal/ventral
axis by regulating target genes such as distalless (dll) and
vestigial (vg) in a concentration-dependent manner
(Neumann and Cohen, 1997b
;
Zecca et al., 1996
). We
investigated Wg signaling in EXT mutant cells by monitoring the distribution
of these proteins.
Levels of Dll protein were slightly reduced in the clones homozygous for
botv423 or ttv524
(Fig. 5B,D). In the cells
mutant for sotv326, the effect was too subtle to assess
definitively, probably because the sotv326 allele is weak,
as described above. The reduction in Dll protein level extended to the
wild-type cells near the clone (Fig.
5C,D, arrows). Furthermore, the Dll protein levels were sometimes
higher at the edge of the clone nearest to the Wg source, as in the case of Hh
and Dpp target genes (Fig.
5B-D). The similar phenotype was also observed for another target,
Vg in botv423 and ttv524 mutant
clones, but too subtle to assess in sotv mutant clones (data not
shown). The previous study did not detect any effects of Ttv on the Wg
signaling (The et al., 1999).
Because the difference could be ascribed to the alleles used, we re-examined
this finding using the same allele ttvl(2)00681 that was
used in the previous study (The et al.,
1999
) or our weak allele ttv205. We found that
in these mutant clones Wg signaling was also slightly decreased. Thus, these
results suggest that Drosophila EXT genes contribute to Wg signaling
in the wing imaginal disc.
|
In the morphogen-expressing region, hh expression was not downregulated, however levels of Hh protein were significantly decreased (Fig. 6A). This may indicate that Hh protein is destabilized and/or not retained efficiently on the cell surface in the absence of HSPGs. In contrast to hh, expression of the wg and dpp and levels of Wg and Dpp were decreased in the EXT clones (Fig. 6B,C). The decrease in dpp expression is easily accountable because Hh signaling is impaired in the absence of HSPGs (Fig. 3). In contrast, the decrease in wg expression is not as readily explainable: cut and wg are both targets of Notch signaling, however the protein level of Cut was not altered in EXT clones (data not shown). This suggests that wg is also regulated by unknown mechanism dependent on HSPGs.
In the morphogen-receiving region, each of these proteins was significantly decreased in the clones of cells mutant for EXT genes (Fig. 6D-F), although a little leakage of morphogen molecules was seen even in the clones doubly mutant for ttv and botv. This suggests two possible mechanisms that do not exclude each other: in the absence of HSPGs these three morphogens are 1) destabilized and/or are not retained efficiently on the cell surface, like Hh in morphogen-expressing region, or 2) prevented from diffusing efficiently into the region consisting of EXT mutant cells. Intriguingly, close observation of the distribution of Hh strongly suggested a function for HSPGs in morphogen movement. In the wild-type discs, Hh protein synthesized in the posterior compartment appears to flow into the anterior compartment, with a moderate concentration gradient starting from the middle of the posterior compartment (Fig. 7A-C). However, Hh abnormally accumulated in the posterior compartment when the EXT mutant clone was in the anterior compartment along the A/P boundary (Fig. 7D-I). This effect was seen both in the ventral region (Fig. 7D-F) and in the dorsal region (Fig. 7G-I). This suggests that Hh failed to move into the mutant cells and as a consequence accumulated in posterior cells instead. Dpp-GFP and Wg accumulation in front of the mutant clones was also apparent, however less pronounced compared with the case of Hh (Fig. 7J-M). Therefore we conclude that the HSPG-dependent diffusion is the common mechanism for the movement of these three morphogens.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
HSPGs are required for Hh, Wg and Dpp signaling
Extensive studies of sgl, sfl and ttv showed that
morphogen signaling and morphogenesis were affected in these HSPG mutants
(reviewed by Lin and Perrimon,
2000; Selleck,
2001
). sgl and sfl mutants had the broadest
effects on signaling, compromising Wg- and Hh-regulated processes. In
contrast, ttv seemed to have affected only Hh signaling, leaving
Wg-mediated patterning unaffected in both embryonic and imaginal development
(Bellaiche et al., 1998
;
The et al., 1999
). These
phenotypes suggested that HSPGs can distinguish between these different
morphogens.
Findings on the three EXT mutants identified in this work, however, argue for a more general role for EXT genes in cell-cell signaling. We found that three EXT family genes, including ttv, affected Hh and Dpp signaling in a similar way. The effect on Wg signaling was not so pronounced compared with that on Hh and Dpp, but the subtle effect on target gene expression and clear reduction in the protein level in the mutant clones also suggest a role for the EXT genes in Wg signaling. The explanation for this contradiction might be that, as seen in Fig. 5, Wg signaling appears to depend on the EXT activity to a lesser degree than Hh and Dpp signaling, so previous study might have missed the role of EXT genes in Wg signaling. We therefore suggest that differences in the behavior of Hh, Wg and Dpp may be more quantitative than qualitative.
ttv and sotv do not encode redundant functions
Although Ttv and Sotv are expected to have similar enzymatic activities,
mutants defective in either of the ttv or sotv genes had
dramatically impaired HSPG biosynthesis and morphogen signaling. Therefore,
despite their similarities, they cannot compensate for each other. They
apparently share this property with their human homologues.
EXT1 and EXT2, the human homologues of ttv and sotv, do
not compensate for each other in vitro or in vivo
(McCormick et al., 2000;
Senay et al., 2000
). In
cultured cells, GFP-tagged EXT1 and EXT2 localize predominantly to the
endoplasmic reticulum when expressed independently. However, GFP-tagged EXT1
and EXT2 formed hetero-oligomeric complexes that accumulated in the Golgi when
expressed together in the same cell. Moreover, the Golgi-localized EXT1-EXT2
complex has substantially higher glycosyltransferase activity than EXT1 or
EXT2 alone, which suggests that this complex represents the biologically
relevant form. These findings provide a rationale to explain how inherited
mutations in either of the two EXT genes can cause loss of activity, resulting
in hereditary multiple extosis. In Drosophila, the observed lack of
complementation is also consistent with an active enzymatic complex that
consists of both Ttv and Sotv.
HSPGs are required for morphogen movement
Gradients are formed by spread of morphogen from a localized source, but
whether this occurs by simple diffusion or by more elaborate mechanisms is not
known. Arguments against morphogen movement by diffusion have been raised by
many, including Kerszberg and Wolpert
(Kerszberg and Wolpert, 1998)
who proposed that morphogens use a `bucket brigade' mechanism in which
receptor-bound morphogen on one cell is transferred to receptors on an
adjacent cell. Alternatively, Entchev et al.
(Entchev et al., 2000
) proposed
transport through `planar transcytosis', a process by which morphogens move by
repeated cycles of endocytosis and exocytosis in the plane of an epithelium.
The notion that Dpp and other morphogens, such as Wg and Hh, all pass through
tissues by transcytosis or similar processes has been championed by many (e.g.
Greco et al., 2001
;
Moline et al., 1999
;
Narayanan and Ramaswami, 2001
;
Pfeiffer and Vincent, 1999
),
albeit not all (McDowell et al.,
2001
; Strigini and Cohen,
2000
) investigators. The third mechanism that has been considered
includes serial passage through neighboring cells through GPI-anchored
proteoglycans (glypicans) (The et al.,
1999
). GPI-linked proteins are inserted in only the outer leaflet
of the plasma membrane, and have been shown to transfer from the plasma
membrane of one cell to another when these cells are in contact, by flip-flap
between adjacent outer leaflets.
We still do not know by which mechanism morphogen diffusion occurs in an intact organism. Our observation that morphogen signaling and protein levels are reduced in the mutant clone, and morphogens accumulated in front of the mutant clones, led us to suppose that HSPGs create an environment that supports the efficient movement of much of the morphogen molecules as scaffolds. However, we cannot exclude the possibility that other systems such as `bucket brigade' or `planar transcytosis' would require the recognition by HSPGs on cell surfaces for cell-to-cell transfer. In addition, we also observed a little leakage of morphogen molecules and signaling in the EXT mutant clones. A possible explanation accounting for these observations might be that in our EXT mutant clones a little amount of HSPGs is still produced, and they barely contribute the morphogen diffusion. Alternatively, other transport mechanisms that do not require HSPGs may play supplementary roles. In either way, all movement of the morphogens must occur within the contiguous plane of this surface environment, because tissues such as wing imaginal discs are folded in numerous places, and no evidence for skipping of morphogens over folds of epithelia was found. More work is necessary to find out how the morphogen gradient is established and maintained in developing organisms.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
Basler, K. and Struhl, G. (1994). Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368,208 -214.[CrossRef][Medline]
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]
Bier, E. (2000). Drawing lines in the Drosophila wing: initiation of wing vein development. Curr. Opin. Genet. Dev. 10,393 -398.[CrossRef][Medline]
Binari, R. C., Staveley, B. E., Johnson, W. A., Godavarti, R.,
Sasisekharan, R. and Manoukian, A. S. (1997). Genetic
evidence that heparin-like glycosaminoglycans are involved in wingless
signaling. Development
124,2623
-2632.
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]
Chen, Y. and Struhl, G. (1996). Dual roles for Patched in sequestering and transducing Hedgehog. Cell 87,553 -563.[Medline]
David, G., Bai, X. M., Van der Schueren, B., Cassiman, J. J. and Van den Berghe, H. (1992). Developmental changes in heparan sulfate expression: in situ detection with mAbs. J. Cell Biol. 119,961 -975.[Abstract]
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., Takeo, S., Kamimura, K., Matsuo, T., Aigaki, T.,
Izumi, S. and Nakato, H. (2003). Dally regulates Dpp
morphogen gradient formation in the Drosophila wing.
Development 130,1515
-1522.
Gerlitz, O. and Basler, K. (2002). Wingful, an
extracellular feedback inhibitor of Wingless. Genes
Dev. 16,1055
-1059.
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]
Greco, V., Hannus, M. and Eaton, S. (2001). Argosomes: a potential vehicle for the spread of morphogens through epithelia. Cell 106,633 -645.[Medline]
Gurdon, J. B., Dyson, S. and St Johnston, D. (1998). Cells' perception of position in a concentration gradient. Cell 95,159 -162.[Medline]
Hacker, U., Lin, X. and Perrimon, N. (1997).
The Drosophila sugarless gene modulates Wingless signaling and
encodes an enzyme involved in polysaccharide biosynthesis.
Development 124,3565
-3573.
Haerry, T. E., Heslip, T. R., Marsh, J. L. and O'Connor, M.
B. (1997). Defects in glucuronate biosynthesis disrupt
Wingless signaling in Drosophila. Development
124,3055
-3064.
Han, C., Lin, X., Zhao, C., Opoka, R., Wang, K. and Lin, X. (2001). Identification of botv, a new segment polarity gene that encodes a protein of the putative tumor suppressor EXT family. In 42nd Annual Drosophila Research Conference (Washington, D.C., Program No. 411).
Han, C., Belenkaya, T., Lin, X., Opoka, R. and Lin, X. (2002). Functional analysis of EXT family of tumor suppressor genes in Drosophila. In 43rd Annual Drosophila Research Conference (San Diego, 2001 Program No. 582C).
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.
Kerszberg, M. and Wolpert, L. (1998). Mechanisms for positional signalling by morphogen transport: a theoretical study. J. Theor. Biol. 191,103 -114.[CrossRef][Medline]
Kim, B. T., Kitagawa, H., Tamura Ji, J., Kusche-Gullberg, M.,
Lindahl, U. and Sugahara, K. (2002). Demonstration of
a novel gene DEXT3 of Drosophila melanogaster as the essential
N-acetylglucosamine transferase in the heparan sulfate biosynthesis: chain
initiation and elongation. J. Biol. Chem.
277,13659
-13665.
Lawrence, P. A. and Struhl, G. (1996). Morphogens, compartments, and pattern: lessons from Drosophila? Cell 85,951 -961.[Medline]
Lee, J. J., von Kessler, D. P., Parks, S. and Beachy, P. A. (1992). Secretion and localized transcription suggest a role in positional signaling for products of the segmentation gene hedgehog.Cell 71,33 -50.[Medline]
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., 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.
McCormick, C., Duncan, G., Goutsos, K. T. and Tufaro, F.
(2000). The putative tumor suppressors EXT1 and EXT2 form a
stable complex that accumulates in the Golgi apparatus and catalyzes the
synthesis of heparan sulfate. Proc. Natl. Acad. Sci.
USA 97,668
-673.
McDowell, N., Gurdon, J. B. and Grainger, D. J. (2001). Formation of a functional morphogen gradient by a passive process in tissue from the early Xenopus embryo. Int. J. Dev. Biol. 45,199 -207.[CrossRef][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.
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.
Narayanan, R. and Ramaswami, M. (2001). Endocytosis in Drosophila: progress, possibilities, prognostications. Exp. Cell Res. 271,28 -35.[CrossRef][Medline]
Neumann, C. and Cohen, S. (1997a). Morphogens and pattern formation. BioEssays 19,721 -729.[Medline]
Neumann, C. J. and Cohen, S. M. (1997b).
Long-range action of Wingless organizes the dorsal-ventral axis of the
Drosophila wing. Development
124,871
-880.
Panganiban, G., Sebring, A., Nagy, L. and Carroll, S. (1995). The development of crustacean limbs and the evolution of arthropods. Science 270,1363 -1366.[Abstract]
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]
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-ß family members is a critical determinant in specifying Smad isoform activation. FEBS Lett. 434,83 -87.[CrossRef][Medline]
Pfeiffer, S. and Vincent, J. P. (1999). Signalling at a distance: transport of Wingless in the embryonic epidermis of Drosophila. Semin. Cell Dev. Biol. 10,303 -309.[CrossRef][Medline]
Porter, J. A., Young, K. E. and Beachy, P. A.
(1996). Cholesterol modification of hedgehog signaling proteins
in animal development. Science
274,255
-259.
Reichsman, F., Smith, L. and Cumberledge, S. (1996). Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction. J. Cell Biol. 135,819 -827.[Abstract]
Selleck, S. B. (2001). Genetic dissection of proteoglycan function in Drosophila and C. elegans. Semin. Cell Dev. Biol. 12,127 -134.[CrossRef][Medline]
Senay, C., Lind, T., Muguruma, K., Tone, Y., Kitagawa, H.,
Sugahara, K., Lidholt, K., Lindahl, U. and Kusche-Gullberg, M.
(2000). The EXT1/EXT2 tumor suppressors: catalytic activities and
role in heparan sulfate biosynthesis. EMBO Rep.
1, 282-286.
Strigini, M. and Cohen, S. M. (1997). A
Hedgehog activity gradient contributes to AP axial patterning of the
Drosophila wing. Development
124,4697
-4705.
Strigini, M. and Cohen, S. M. (2000). Wingless gradient formation in the Drosophila wing. Curr. Biol. 10,293 -300.[CrossRef][Medline]
Sturtevant, M. A., Biehs, B., Marin, E. and Bier, E.
(1997). The spalt gene links the A/P compartment
boundary to a linear adult structure in the Drosophila wing.
Development 124,21
-32.
Sugahara, K. and Kitagawa, H. (2000). Recent advances in the study of the biosynthesis and functions of sulfated glycosaminoglycans. Curr. Opin. Struct. Biol. 10,518 -527.[CrossRef][Medline]
Tabata, T. (2001). Genetics of morphogen gradients. Nat. Rev. Genet. 2, 620-630.[CrossRef][Medline]
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]
Taipale, J. and Keski-Oja, J. (1997). Growth
factors in the extracellular matrix. FASEB J.
11, 51-59.
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., 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]
Willert, K., Brown, J. D., Danenberg, E., Duncan, A. W., Weissman, I. L., Reya, T., Yates, J. R., 3rd and Nusse, R. (2003). Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423,448 -452.[CrossRef][Medline]
Wolpert, L. (1969). Positional information and the spatial pattern of cellular differentiation. J. Theor. Biol. 25,1 -47.[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.
Zecca, M., Basler, K. and Struhl, G. (1996). Direct and long-range action of a Wingless morphogen gradient. Cell 87,833 -844.[Medline]