1 PRESTO, Japan Science and Technology Corporation
2 Department of Biological Science and Technology, Tokyo University of Science,
Yamazaki 2641, Noda, Chiba 278-8510, Japan
3 Genome and Drug Research Center, Tokyo University of Science, Yamazaki 2641,
Noda, Chiba 278-8510, Japan
4 Department of Nutrition, School of Medicine, University of Tokushima, 3-18-15
Kuramoto, Tokushima 770-8503, Japan
5 Department of Molecular and Tumor Pathology, Chiba University Graduate School
of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan
6 Department of Biology, Developmental, Cell and Molecular Biology Group, Duke
University, Durham, NC 27708, USA
7 Department of Genetics, Howard Hughes Medical Institute, Harvard Medical
School, 200 Longwood Avenue, Boston, MA 02115, USA
* Author for correspondence (e-mail: matsuno{at}rs.noda.tus.ac.jp)
Accepted 20 June 2003
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SUMMARY |
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Key words: Notch signalling, Neurogenic gene, neurotic, nti, O-fucosyltransferase, Fucose, Fringe, Notch-Delta binding, Drosophila
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Introduction |
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Deficiency of essential genes for Notch signalling causes overproduction of
neurons in the Drosophila embryo: the `neurogenic' phenotype
(Campos-Ortega, 1995). Some
essential components of Notch signalling are associated with the neurogenic
phenotype when both the zygotic genes and maternally contributed mRNA are
abolished. These genes are referred to as `maternal neurogenic genes'. Several
genes relatively recently identified as components of Notch signalling, such
as Suppressor of Hairless, Kuzbanian, Presenilin and Nicastrin belong to this
class of genes (Lecourtois and
Schweisguth, 1995
; Rooke et
al., 1996
; Struhl and
Greenwald, 1999
; Ye et al.,
1999
; Chung and Struhl,
2001
; López-Schier and
St Johnston, 2002
; Hu et al.,
2002
). Because of the difficulty to screen for maternal
phenotypes, not all maternal neurogenic genes have yet been identified. We
report a novel maternal neurogenic gene, neurotic (nti;
O-fut1 FlyBase), which encodes the
O-fucosyltransferase. The activity of nti is also needed in
wing margin formation, indicating that nti is an essential component
of Notch signalling. Epistatic analysis showed that nti is essential
for full-length Notch and Fng function but not for NotchICD. We
also show that Nti is essential for the physical interaction between Notch and
Delta in Drosophila cultured cells. Thus, our results establish
Neurotic/OFUT1 as a moderator of Notch-ligand interactions, which has both
Fng-dependent and Fng-independent functions.
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Materials and methods |
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Genetics
To generate mosaic clones, larvae were heat-shocked at 37°C for 1 hour
48-72 hours (germline mosaics, Fig.
3B,D) or 24-48 hours (others) after egg laying. Embryos that lack
maternal nti function were laid by y w hs-flp/+;
FRTG13 ovoD1/FRTG13 nti
female. These females were crossed with FRTG13
nti/CyO, ftz-lacZ males. Somatic clones were made in y w
hs-flp; FRTG13 nti/FRTG13 Ubi-GFP
larvae, and for rescue experiment, y w hs-flp; FRTG13
nti/FRTG13 Ubi-GFP; hs-nti/+ larvae
were used.
|
Immunohistochemistry
Wing imaginal discs dissected from late third instar larvae were
immunostained as previously described
(Matsuno et al., 2002).
Immunostaining and in situ hybridisation to whole-mount embryos were performed
according to standard protocols. Antibodies used for immunostaining were
anti-Elav (9F8A9, dilution 1:10) (O'Neill
et al., 1994
), anti-Wg (4D4, 1:500)
(Brook and Cohen, 1996
), and
anti-Notch (C17.9C6, 1:500) (Fehon et al.,
1990
). For observation, fluorescent microscopy
(Fig. 1G,H) and confocal
microscopy (BioRad Radience 2100, Fig.
1A-D, Fig. 2D,
Fig. 3) were used.
|
|
Western blotting
Transfections were done as additional replicates for the binding assay.
Protein was extracted from the cells with 20 mM HEPES-NaOH (pH 7.9) buffer
containing 0.5% NP-40, 15% glycerol, 300 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM
dithiothreitol, 1 mM sodium orthovanadate and proteinase inhibitors
(Kitagawa et al., 2001). After
separation by SDS-polyacrylamide electrophoresis, proteins were
electrophoretically transferred onto PVDF membranes (BioRad). Primary
antibodies used for blotting were anti-Notch intracellular domain (C17.9C6),
anti-Myc (9E10), anti-HA (12CA5) and anti-tubulin (E7)
(Chu and Klymkowsky, 1989
).
They were visualised with appropriate secondary antibodies conjugated with
horseradish peroxidase, ECL (Amersham Pharmacia Biotech) chemiluminescent
substrate and X-ray films. In some cases, the membranes were stripped and
reblotted.
Flow cytometric analysis
S2 cells were transfected with pRmHa-3-based expression vectors for GFP
(gift from R. Tsuda), Notch and nti RNAi as described above. Cells
mechanically detached from tissue culture dishes were washed in
phosphate-buffered saline (PBS) once and incubated with an anti-Notch
extracellular domain antibody (Rat-1, gift from S. Artavanis-Tsakonas) at
4°C for 20 minutes. After washing with PBS, the cells were incubated with
a phycoerythrin-labeled anti-rat IgG antibody for 20 minutes at 4°C,
washed with PBS and analysed with a FACScan (BD Biosciences).
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Results |
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neurotic encodes a O-fucosyltransferase homolog
The nti mutation 4R6 was mapped to 50C-D by complementation tests
to deficiency lines, and nucleotide sequence analysis of the mutant chromosome
revealed a nonsense mutation in the annotated gene CG12366. The single
mutation changes the Lysine at position 133 in the putative ORF of 402 amino
acids to a premature stop codon (AAG to TAG;
Fig. 2A), strongly suggesting
that 4R6 causes complete loss of CG12366 function. As expected from the
nti maternal effect, CG12366 mRNA is uniformly distributed in early
embryos but decreases during later stages
(Fig. 2B,C; data not shown). To
confirm that the nti mutant phenotype is caused by a disruption in
CG12366, we tested whether inducible expression of CG12366 could rescue
nti mutant cells. As shown in Fig.
2D, Wg expression in the nti clones is rescued in wing
imaginal discs by ubiquitous expression of CG12366 (hs-nti). The
maternal neurogenic phenotype could also be partially rescued using
hs-nti (data not shown). We also constructed a hairpin construct
(nti-IR) to express in vivo a dsRNA that would inhibit CG12366
function. Expression of nti-IR using ptc-Gal4 causes wing
nicks reminiscent of loss of Notch activity
(de Celis and Garcia-Bellido,
1994) and nti mutant clones, consistent with
RNAi-mediated inactivation of nti. We used this nti-IR
expression construct to knock down nti function in cultured cells
(see below). These results demonstrate that CG12366 encodes nti
function.
Database searches revealed that nti encodes a putative protein
with significant homology to human GDP-fucose O-fucosyltransferase 1
(Wang et al., 2001)
(O-FucT-1; Fig. 2A).
O-FucT-1 is an enzyme that adds a carbohydrate, fucose, to serine or
threonine residues in the consensus sequence CXXGGS/TC (X is
any amino acids) between the second and third conserved cysteines of EGF
repeats (Harris and Spellman,
1993
; Wang et al.,
2001
). Notch and its ligands, Delta and Serrate, have such
sequences in their extracellular EGF repeats, and all of them are
O-fucosylated (Moloney et al.,
2000a
; Moloney et al.,
2000b
; Panin et al.,
2002
). Recently, Okajima and Irvine
(Okajima and Irvine, 2002
)
have demonstrated that Drosophila O-FucT-1 is required for the
activation of Notch by its ligands using a RNAi knockdown approach.
Furthermore, Shi and Stanley (Shi and
Stanley, 2003
) showed that O-FucT-1 mutant mice have a
phenotype similar to that of Presenilins or RBP-J
. Thus our present
results confirm these previous observations and support the model that
O-FucT-1 is an essential component of Notch signalling.
Fringe (Fng), a modulator of Notch signalling, adds
N-acetylglucosamine (GlcNAc) onto the O-fucose moieties of
Notch and its ligands (Brückner et
al., 2000; Moloney et al.,
2000a
). Modification of Notch by Fng, and subsequent glycosylation
of Notch, modulates the binding of Delta and Serrate to Notch
(Fleming et al., 1997
;
Panin et al., 1997
;
Moloney et al., 2000a
;
Brückner et al., 2000
;
Chen et al., 2001
). These
reports and our results suggest that nti enables Notch to respond to
its ligands by adding O-linked fucose to the Notch EGF repeats.
However, our finding may also be unexpected because no report has ever been
associated fng mutations with the neurogenic phenotype
(Irvine, 1999
). Therefore,
nti, like Notch, is essential for lateral inhibition during
neuroblast segregation, a process for which fng is probably
dispensable. Furthermore, the nti mutant phenotype in adult wings is
also distinct from that of fng
(Irvine, 1999
). These results
indicate that nti has a function that is independent of fng.
Such a fng-independent function was also proposed from the analysis
of the neurogenic phenotype induced by RNAi of Nti/OFUT1 in
Drosophila bristles (Okajima and
Irvine, 2002
).
Neurotic functions between full-length Notch and NotchICD,
and is essential for Fng function
To define precisely the function of nti in the Notch signalling
cascade, we investigated the epistatic relationship between nti and
other genes involved in Notch signalling. We first asked whether the activity
of the Notch intracellular domain (NotchICD), which acts as a
constitutively active Notch receptor
(Struhl et al., 1993),
requires nti. As shown in Fig.
3A, expression of Wg is absent in nti mutant clones.
However, NotchICD could induce Wg expression in nti mutant
clones (Fig. 3B) as it can in
wild-type cells (Diaz-Benjumea and Cohen,
1995
). A similar experiment was carried out with an overexpressed
full-length Notch receptor, which in wild type also induces ectopic Wg
(Fig. 3C)
(Matsuno et al., 2002
).
Interestingly, in contrast to NotchICD, the full-length Notch could
not induce ectopic Wg expression in nti mutant clones
(Fig. 3D), indicating that
nti is required for the activity of full-length Notch but not for its
intracellular domain. These interactions are consistent with the idea that Nti
modifies the extracellular domain of Notch or its ligands.
Next, we investigated the epistatic relationship between nti and
fng. From the structure of the O-linked tetrasaccharide
attached to the EGF repeats of Notch and from enzymatic functions of Nti and
Fng, it was speculated that Nti activity is a prerequisite for the function of
Fng (Brückner et al.,
2000; Moloney et al.,
2000a
). As previously reported, ectopic expression of Fng in the
ventral compartment of the wing imaginal disc induced Wg ectopically
(Fig. 3E)
(Panin et al., 1997
). By
contrast, ectopic expression of Fng in nti mutant clones did not
induce ectopic expression of Wg, and endogeneous expression of Wg is absent in
these clones (Fig. 3F). These
results indicate that Nti is essential for Fng function. These findings are
consistent with the RNAi experiments against Nti/OFUT1
(Okajima and Irvine,
2002
).
Neurotic is essential for Delta-Notch binding
As both Notch and its ligands are O-fucosylated, we next analysed
the effects of nti expression on the ligand-receptor interaction
using the method of Brückner et al.
(Brückner et al., 2000).
S2 cultured cells were transfected with Notch and subsequently incubated with
conditioned medium containing a Delta-alkaline phosphatase fusion protein
(Delta-AP) prepared also by transfection to S2 cells. As S2 cells are
Notch-deficient (Fehon et al.,
1990
), AP activity specifically bound to Notch-transfected cells
will reflect the ligand binding activity of Notch. As previously reported,
co-expression of Fng with Notch increases its ability to bind to Delta
(Fig. 4A, compare lanes 2,3)
(Brückner et al., 2000
).
Nti co-expression alone does not significantly alter the binding ability of
Notch (Fig. 4A, lane 4). We
speculated that Nti, expressed endogenously, is saturated for the Notch-Delta
binding under this condition. Endogenous activity of Nti/OFUT1 in S2 cells was
reported previously (Okajima and Irvine,
2002
). However, co-expression of Nti with Fng potentiates Notch
binding activity (Fig. 4A,
lanes 5,7). Importantly, co-expression of Nti or Fng does not significantly
change the expression of Notch protein in the cells
(Fig. 4A, Anti-Notch). We noted
that co-expression of Nti-Myc, but not wild-type Nti, with Notch inhibited the
Notch-Delta binding, suggesting that Nti-Myc behaves as a dominant-negative
protein (Fig. 4A, lane 6, see
Discussion).
|
To test the possibility that nti might affect Notch presentation
on the cell surface, we examined expression of Notch in live transfected cells
using flow cytometry. In this experiment, S2 cells were co-transfected with
GFP and Notch expression constructs, as well as either nti, nti-IR or
control constructs, and stained with an antibody raised against the Notch
extracellular domain. As shown in Fig.
4C, the ratio of double positive cells for GFP and the anti-Notch
(cells expressing Notch on the cell surface) was not significantly affected by
either the knockdown or the overexpression of Nti (see upper-right area of
each). Thus, Notch is expressed in a form accessible to the antibody and
probably to the ligand irrespective of nti activity. These results
suggest that Nti affects the physical interaction between Notch and Delta,
rather than cellular distribution or transport of Notch. It has been reported
that Delta and Serrate are also O-fucosylated
(Panin et al., 2002). However,
neither knockdown nor the overexpression of Nti affects the ability of Delta
to bind Notch (Fig. 5). These
results are consistent with the in vivo result that nti functions
cell autonomously (Fig.
3A).
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Discussion |
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Notch signalling is involved in two major classes of cell-cell signalling:
lateral inhibition and inductive signalling
(Artavanis-Tsakonas et al.,
1999). Neuroblast segregation in early embryogenesis is a well
known example of lateral inhibition, and failure of the Notch signalling
pathway during this process leads to an hypertrophy of neuroblasts, known as
the neurogenic phenotype (Campos-Ortega,
1995
). However, induction of several genes, such as wg,
along the dorsal/ventral compartmental boundary of the wing disc depends on
inductive Notch signalling. In both classes of Notch signalling events, the
Notch and nti mutant phenotypes are identical, indicating
that Nti is essential for Notch signalling. Our finding differs with the
recent report using nti RNAi
(Okajima and Irvine, 2002
), as
apparent embryonic neurogenic phenotypes were not noted. This discrepancy is
probably due to the partial knockdown effect generated by RNAi.
There are a considerable number of genes, including the ligands of the
Notch receptor, that encode putative proteins that contain EGF motifs with
consensus sequences for O-fucosylation in the Drosophila
genome (Haltiwanger, 2002).
However, mutant phenotypes of nti are strikingly similar to those of
the Notch receptor. In fact, in addition to the wing phenotype shown above,
nti somatic clones exhibit mutant phenotypes that are characteristic
of Notch loss-of-function mutations. These include defects on the notum
bristles, rough eye and the leg segment fusion (data not shown). These
phenotypes were also observed in the knockdown study of nti
(Okajima and Irvine, 2002
).
These results indicate that the primary target of O-fucosylation of
Nti is Notch, while there may be unrevealed signalling pathways that
nti function is required. Roos et al.
(Roos et al., 2002
) have
reported that there is another putative O-fucosyltransferase in
Drosophila and mammals. Thus, it is tempting to speculate that each
O-fucosyltransferase has distinct target(s) that are involved in
specific cell signalling pathways.
Neurotic has both Fng-dependent and Fng-independent function in Notch
signalling
As O-fucose on Notch has been shown to act as a molecular scaffold
for GlcNAc that is elongated by Fng, one would expect that the phenotypes of
O-fucosyltransferase mutant might be the same as those of
fng. Unexpectedly, however, we found that the nti and
fng mutant phenotypes are quite different. Strikingly, the embryonic
neurogenic phenotype that is evident in nti mutant, and is an
indication of its essential role in Notch signalling, has never been reported
in fng mutants (Irvine,
1999). Furthermore, it is thought that Fng does not have a
significant role in lateral inhibition, while it is involved in generation of
the cell boundary between cells expressing Fng and cells not expressing Fng
(Irvine, 1999
). Additionally,
our in vitro binding assay revealed that Nti is essential for binding between
Notch and Delta. Based on the previous findings and our present results, we
propose that O-glycosylation of Notch EGF repeats has two distinct
roles for binding to Delta (Fig.
7). First, O-fucosylation catalysed by Nti is an absolute
requirement for binding between Notch and the ligand, and this binding is
sufficient to accomplish lateral inhibition. For this function, no additional
glycosylation to O-fucose residue is required. This idea is also
supported by the observation that in the tissues and organisms that do not
express fng, Delta is competent to activate the Notch receptor
(Panin et al., 1997
). In this
respect, it is worth noting that in the C. elegans genome there is a
highly conserved nti, while a fng homolog is not found
(Wang et al., 2001
). Second,
addition of GlcNAc to the O-fucose residue by Fng enhances the
interactions between Notch and Delta, modulating the receptor-ligand
interactions (Fleming et al.,
1997
; Panin et al.,
1997
; Klein and Arias,
1998
). In fact, it is known that the expression of fng
has high degree of regional specificity, and the boundary of the cells
expressing and not expressing Fng often defines the border of distinct tissue
structures (Irvine and Wieschaus,
1994
; Dominguez and de Celis,
1998
; Cho and Choi,
1998
). Thus, the region-specific expression of fng allows
modulation of Notch signalling, resulting in generation of complex structure
of organs. As expected from the second function of Nti, its function is
essential for Fng-dependent modulation of Notch signalling as well as
Fng-independent function. In the wing disc, nti is epistatic to
fng, and fng requires nti to induce Wg at the
dorsal and ventral compartment boundary. Additionally, in in vitro binding
assay, Fng depends on Nti to enhance the binding between Notch and Delta.
These lines of evidence indicate that Nti is involved in Fng-dependent
modulation of Notch signalling, which is consistent with an O-glycan
structure of the Notch EGF repeats
(Brückner et al., 2000
;
Moloney et al., 2000a
). This
Fng-dependent function of Nti is not contradictory to our first model.
|
In this work, we failed to demonstrate an effect of nti on
Notch-Serrate binding, because, as reported before, Notch-Serrate-AP binding
could not be detected by this binding assay
(Brückner et al., 2000).
However, phenotypes observed in the somatic clones of nti generated
in the wing disc strongly suggests that Nti affects the Notch-Serrate binding
(Fig. 1H). For example, in the
nti clones near the dorsal and ventral boundary from the ventral
side, induction of Wg is impaired, indicating that nti clones fail to
receive the Serrate signal. Thus, we propose that the requirement of
nti for Notch-ligand binding is not restricted to Delta. However, the
neurogenic phenotype of nti could be wholly explained by the effect
on Notch-Delta binding, as Delta is the only the ligand of Notch involved in
neuroblast segregation during early embryogenesis
(Thomas et al., 1991
).
In our Notch-Delta binding assay, co-expression of Nti and Fng with Notch
increased the Notch-Delta binding over that of Fng with Notch, whereas
co-expression of a wild-type Nti with Notch did not increase this binding. We
speculate that the amount of Nti endogenously expressed in S2 cells, as
evidenced by the effectiveness of nti-IR shown in
Fig. 4B, saturates the ability
of fucose alone to promote the ligand binding. Indeed, it was shown that S2
cells endogenously expressed Nti/OFUT1
(Okajima and Irvine, 2002).
However it does not imply that addition of O-fucose to Notch by Nti
is also saturated under these conditions. Namely, the increase of
O-fucosyltransferase activity, which does not lead to elevation of
Delta binding per se, could increase the Notch-Delta binding after elongation
of the O-fucose residue by Fng. These results suggest that linear
range by which fucose-GlcNAc moiety can elevate the ligand binding is wider
than that by which fucose alone can. Multiple sites that are possibly
O-fucosylated were found in the EGF repeats of Notch
(Okajima and Irvine, 2002
).
Thus, it is possible that some of these potential O-fucosylation
sites may influence the Delta binding only after modification by Fng. We also
noticed that Nti-myc probably has a dominant-negative function
(Fig. 4A, lane 6). This
dominant-negative activity of Nti-myc is apparently restored by co-expression
with Fng, which makes the ligand binding well over that by Fng expression
alone (Fig. 4A, lanes 3,7).
This is in contrast with the fact that the decreased Notch-Delta binding
associated with RNAi of nti, which is supposed to merely reduce the
endogenous Nti protein, hardly restored by co-expression with Fng
(Fig. 4B). Therefore, we
speculate that the Myc-tag does not simply disrupt the enzymatic activity of
Nti-myc. Some kind of competition among Nti-myc, endogenous Nti, Fng and Notch
may be involved in this dominant-negative effect. In support of this model,
physical interaction between Notch and Fng has been reported
(Ju et al., 2000
). This
dominant-negative effect may also account for the reduction of the Notch-Delta
binding associated with Nti-G3-myc. We speculate that Ntimyc and Nti-G3-myc,
both of which carry the same Myc-tag, have similar dominant negative effects,
which do not depend on the enzymatic activity of O-fucosyltransferase
and which are restored by co-expressing Fng. The slight reduction of the
Notch-Delta binding associated with Nti-G3-myc and Fng over that of Fng alone
may also be caused by this dominant-negative effect of Nti-G3-myc.
Role of O-fucosylation in cell signalling
The importance of glycoconjugates in cell adhesion and cell-cell
communication has been believed for many years, since nearly all cells were
found to be covered with numerous carbohydrate-rich molecules
(Roseman, 2001). More
recently, gene disruption of a number of glycosyltransferases resulted in
embryonic lethality in mice, underscoring their importance in development of
multicellular organisms. However, most of the reports are only descriptive of
the phenotypes and the molecular mechanisms are still elusive
(Furukawa et al., 2001
;
Dennis et al., 1999
). Here, we
have presented one of the very few examples of molecular explanation for such
mechanisms of action. Furthermore, it is an unprecedented example of an
absolute requirement of a protein glycosylation event for a ligand-receptor
interaction. The simplest interpretation of these phenomena would be that
association of Delta to its receptor involves lectin-like protein-carbohydrate
interaction. This hypothesis is consistent with the result that
O-fucosylation induces little conformational change in EGF repeat of
factor VII (Kao et al.,
1999
).
The first protein shown to be O-fucosylated was urinary type
plasminogen activator (uPA) (Kentzer et
al., 1990), followed by tissue-type plasminogen activators and
several clotting factors. It has been shown that O-fucosylation of
uPA is essential for activation of the uPA receptor
(Rabbani et al., 1992
) that
has diverse functions including plasminogen activation
(Dear and Medcalf, 1998
),
although uPA receptor-deficient mice survive to adulthood with no overt
phenotypic abnormalities and are fertile
(Bugge et al., 1995
). Another
O-fucosylated protein is Cripto, a member of the EGF-CFC family.
Cripto is thought to act as an essential co-factor for Nodal signalling
(Minchiotti et al., 2002
).
Amino acid substitution of threonine to alanine, which prevents
O-fucosylation reduced the signalling activity of Nodal, suggesting
that O-fucosylation is important for Cripto function
(Schiffer et al., 2001
;
Yan et al., 2002
). Until now,
more than ten proteins have been found to carry the consensus sequence of
O-fucosylation site (Haltiwanger,
2002
). In this paper, we have shown that O-fucosylation
is mandatory for Notch signalling. Although the O-fucosyltransferase
presented here has remarkable functional specificity to the Notch receptor,
there seems to be plurality of such enzymes
(Roos et al., 2002
).
Therefore, the regulation of protein activity or function by
O-fucosylation could be a general mechanism for many signal
transduction systems.
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ACKNOWLEDGMENTS |
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REFERENCES |
---|
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---|
Artavanis-Tsakonas, S., Matsuno, K. and Fortini, M. E. (1995). Notch signaling. Science 268,225 -232.[Medline]
Artavanis-Tsakonas, S., Rand, M. D. and Lake, R. J.
(1999). Notch signaling: cell fate control and signal integration
in development. Science
284,770
-776.
Bailey, A. M. and Posakony, J. W. (1995). Suppressor of hairless directly activates transcription of enhancer of split complex genes in response to Notch receptor activity. Genes Dev. 9,2609 -2622.[Abstract]
Brook, W. J. and Cohen, S. M. (1996). Antagonistic interactions between wingless and decapentaplegic responsible for dorsal-ventral pattern in the Drosophila leg. Science 273,1373 -1377.[Abstract]
Brückner, K., Perez, L., Clausen, H. and Cohen, S. (2000). Glycosyltransferase activity of Fringe modulates Notch-Delta interactions. Nature 406,411 -415.[CrossRef][Medline]
Bugge, T. H., Suh, T. T., Flick, M. J., Daugherty, C. C., Romer,
J., Solberg, H., Ellis, V., Dano, K. and Degen, J. L. (1995).
The receptor for urokinase-type plasminogen activator is not essential for
mouse development or fertility. J. Biol. Chem.
270,16886
-16894.
Campos-Ortega, J. A. (1995). Genetic mechanisms of early neurogenesis in Drosophila melanogaster. Mol. Neurobiol. 10,75 -89.[Medline]
Chen, J., Moloney, D. J. and Stanley, P.
(2001). Fringe modulation of Jagged1-induced Notch signaling
requires the action of ß4galactosyltransferase-1. Proc. Natl.
Acad. Sci. USA 98,13716
-13721.
Cho, K. O. and Choi, K. W. (1998). Fringe is essential for mirror symmetry and morphogenesis in the Drosophila eye. Nature 396,272 -276.[CrossRef][Medline]
Chou, T. B. and Perrimon, N. (1996). The
autosomal FLP-DFS technique for generating germline mosaics in Drosophila
melanogaster. Genetics 144,1673
-1679.
Chu, D. T. and Klymkowsky, M. W. (1989). The appearance of acetylated alpha-tubulin during early development and cellular differentiation in Xenopus. Dev. Biol. 136,104 -117.[Medline]
Chung, H. M. and Struhl, G. (2001). Nicastrin is required for Presenilinmediated transmembrane cleavage in Drosophila.Nat. Cell Biol. 3,1129 -1132.[CrossRef][Medline]
de Celis, J. F. and Garcia-Bellido, A. (1994). Roles of the Notch gene in Drosophila wing morphogenesis. Mech. Dev. 46,109 -122.[CrossRef][Medline]
de Celis, J. F., Garcia-Bellido, A. and Bray, S. J.
(1996). Activation and function of Notch at the
dorsal-ventral boundary of the wing imaginal disc.
Development 122,359
-369.
Dear, A. E. and Medcalf, R. L. (1998). The urokinase-type-plasminogenactivator receptor (CD87) is a pleiotropic molecule. Eur. J. Biochem. 252,185 -193.[Abstract]
Dennis, J. W., Granovsky, M. and Warren, C. E. (1999). Protein glycosylation in development and disease. BioEssays 21,412 -421.[CrossRef][Medline]
Diaz-Benjumea, F. J. and Cohen, S. M. (1995).
Serrate signals through Notch to establish a Wingless-dependent organizer at
the dorsal/ventral compartment boundary of the Drosophila wing.
Development 121,4215
-4225.
Dominguez, M. and de Celis, J. F. (1998). A dorsal/ventral boundary established by Notch controls growth and polarity in the Drosophila eye. Nature 396,276 -278.[CrossRef][Medline]
Fehon, R. G., Kooh, P. J., Rebay, I., Regan, C. L., Xu, T., Muskavitch, M. A. and Artavanis-Tsakonas, S. (1990). Molecular interactions between the protein products of the neurogenic loci Notch and Delta, two EGF-homologous genes in Drosophila. Cell 61,523 -534.[Medline]
Fleming, R. J., Gu, Y. and Hukriede, N. A.
(1997). Serrate-mediated activation of Notch is
specifically blocked by the product of the gene fringe in the dorsal
compartment of the Drosophila wing imaginal disc.
Development 124,2973
-2981.
Furukawa, K., Takamiya, K., Okada, M., Inoue, M., Fukumoto, S. and Furukawa, K. (2001). Novel functions of complex carbohydrates elucidated by the mutant mice of glycosyltransferase genes. Biochim. Biophys. Acta. 1525, 1-12.[Medline]
Go, M. J., Eastman, D. S. and Artavanis-Tsakonas, S.
(1998). Cell proliferation control by Notch signaling in
Drosophila development. Development
125,2031
-2040.
Greenwald, I. (1998). LIN-12/Notch signaling:
lessons from worms and flies. Genes Dev.
12,1751
-1762.
Gu, Y., Hukriede, N. A. and Fleming, R. J.
(1995). Serrate expression can functionally replace
Delta activity during neuroblast segregation in the
Drosophila embryo. Development
121,855
-865.
Haltiwanger, R. S. (2002). Regulation of signal transduction pathways in development by glycosylation. Curr. Opin. Struct. Biol. 12,593 -598.[CrossRef][Medline]
Harris, R. J. and Spellman, M. W. (1993). O-linked fucose and other post-translational modifications unique to EGF modules. Glycobiology 3,219 -224.[Abstract]
Hinz, U., Giebel, B. and Campos-Ortega, J. A. (1994). The basic-helix-loop-helix domain of Drosophila lethal of scute protein is sufficient for proneural function and activates neurogenic genes. Cell 76,77 -87.[Medline]
Hu, Y., Ye, Y. and Fortini, M. E. (2002).
Nicastrin is required for -secretase cleavage of the
Drosophila Notch receptor. Dev. Cell
2, 69-78.[Medline]
Irvine, K. D. (1999). Fringe, Notch, and making developmental boundaries. Curr. Opin. Genet. Dev. 9, 434-441.[CrossRef][Medline]
Irvine, K. D. and Wieschaus, E. (1994). fringe, a Boundary-specific signaling molecule, mediates interactions between dorsal and ventral cells during Drosophila wing development. Cell 79,595 -606.[Medline]
Ju, B. G., Jeong, S., Bae, E., Hyun, S., Carroll, S. B., Yim, J. and Kim, J. (2000) Fringe forms a complex with Notch. Nature 405,191 -195[CrossRef][Medline]
Kao, Y. H., Lee, G. F., Wang, Y., Starovasnik, M. A., Kelley, R. F., Spellman, M. W. and Lerner, L. (1999). The effect of O-fucosylation on the first EGF-like domain from human blood coagulation factor VII. Biochemistry 38,7097 -7110.[CrossRef][Medline]
Kentzer, E. J., Buko, A. M., Menon, G. and Sarin, V. K. (1990). Carbohydrate composition and presence of a fucose-protein linkage in recombinant human pro-urokinase. Biochem. Biophys. Res. Commun. 171,401 -406.[Medline]
Kidd, S., Lockett, T. J. and Young, M. W. (1983). The Notch locus of Drosophila melanogaster. Cell 34,421 -433.[Medline]
Kitagawa, M., Oyama, T., Kawashima, T., Yedvobnick, B., Kumar,
A., Matsuno, K. and Harigaya, K. (2001). A human protein with
sequence similarity to Drosophila mastermind coordinates the nuclear
form of notch and a CSL protein to build a transcriptional activator complex
on target promoters. Mol. Cell. Biol.
21,4337
-4346.
Klein, T. and Arias, A. M. (1998). Interactions
among Delta, Serrate and Fringe modulate Notch activity during
Drosophila wing development. Development
125,2951
-2962.
Lecourtois, M. and Schweisguth, F. (1995). The neurogenic suppressor of hairless DNA-binding protein mediates the transcriptional activation of the enhancer of split complex genes triggered by Notch signaling. Genes Dev. 9,2598 -2608.[Abstract]
Lee, T. and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis. Neuron 22,451 -461.[Medline]
López-Schier, H. and St Johnston, D. (2002). Drosophila nicastrin is essential for the intramembranous cleavage of notch. Dev. Cell 2, 79-89.[Medline]
Matsuno, K., Ito, M., Hori, K., Miyashita, F., Suzuki, S., Kishi, N., Artavanis-Tsakonas, S. and Okano, H. (2002). Involvement of a proline-rich motif and RING-H2 finger of Deltex in the regulation of Notch signaling. Development 129,1049 -1059.[Medline]
Minchiotti, G., Parisi, S., Liguori, G. L., D'Andrea, D. and Persico, M. G. (2002). Role of the EGF-CFC gene cripto in cell differentiation and embryo development. Gene 287, 33-37.[CrossRef][Medline]
Moloney, D. J., Panin, V. M., Johnston, S. H., Chen, J., Shao, L., Wilson, R., Wang, Y., Stanley, P., Irvine, K. D., Haltiwanger, R. S. and Vogt, T. F. (2000a). Fringe is a glycosyltransferase that modifies Notch. Nature 406,369 -375.[CrossRef][Medline]
Moloney, D. J., Shair, L. H., Lu, F. M., Xia, J., Locke, R.,
Matta, K. L. and Haltiwanger, R. S. (2000b). Mammalian Notch1
is modified with two unusual forms of O-linked glycosylation found on
epidermal growth factor-like modules. J. Biol. Chem.
275,9604
-9611.
Mumm, J. S. and Kopan, R. (2000). Notch signaling: from the outside in. Dev. Biol. 228,151 -165.[CrossRef][Medline]
Okajima, T. and Irvine, K. D. (2002). Regulation of Notch signaling by O-linked fucose. Cell 111,893 -904.[Medline]
O'Neill, E. M., Rebay, I., Tjian, R. and Rubin, G. M. (1994). The activities of two Ets-related transcription factors required for Drosophila eye development are modulated by the Ras/MAPK pathway. Cell 78,137 -147.[Medline]
Panin, V. M., Papayannopoulos, V., Wilson, R. and Irvine, K. D. (1997). Fringe modulates Notch-ligand interactions. Nature 387,908 -912.[CrossRef][Medline]
Panin, V. M., Shao, L., Lei, L., Moloney, D. J., Irvine, K. D.
and Haltiwanger, R. S. (2002). Notch ligands are substrates
for protein O-fucosyltransferase-1 and Fringe. J. Biol.
Chem. 277,29945
-29952.
Rabbani, S. A., Mazar, A. P., Bernier, S. M., Haq, M., Bolivar,
I., Henkin, J. and Goltzman, D. (1992). Structural
requirements for the growth factor activity of the amino-terminal domain of
urokinase. J. Biol. Chem.
267,14151
-14156.
Rebay, I., Fleming, R. J., Fehon, R. G., Cherbas, L., Cherbas, P. and Artavanis-Tsakonas, S. (1991). Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor. Cell 67,687 -699.[Medline]
Robinow, S. and White, K. (1988). The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages. Dev. Biol. 126,294 -303.[Medline]
Rooke, J., Pan, D., Xu, T. and Rubin, G. M. (1996). KUZ, a conserved metalloprotease-disintegrin protein with two roles in Drosophila neurogenesis. Science 273,1227 -1231.[Abstract]
Roos, C., Kolmer, M., Mattila, P. and Renkonen, R.
(2002). Composition of Drosophila melanogaster proteome
involved in fucosylated glycan metabolism. J. Biol.
Chem. 277,3168
-3175.
Roseman, S. (2001). Reflections on
glycobiology. J. Biol. Chem.
276,41527
-41542.
Rulifson, E. J. and Blair, S. S. (1995).
Notch regulates wingless expression and is not required for
reception of the paracrine wingless signal during wing margin
neurogenesis in Drosophila. Development
121,2813
-2824.
Schiffer, S. G., Foley, S., Kaffashan, A., Hronowski, X.,
Zichittella, A. E., Yeo, C. Y., Miatkowski, K., Adkins, H. B., Damon, B.,
Whitman, M., Salomon, D., Sanicola, M. and Williams, K. P.
(2001). Fucosylation of Cripto is required for its ability to
facilitate nodal signaling. J. Biol. Chem.
276,37769
-37778.
Shi, S. and Stanley, P. (2003). Protein
O-fucosyltransferase 1 is an essential component of Notch signaling
pathways. Proc. Natl. Acad. Sci. USA
100,5234
-5239.
Struhl, G. and Adachi, A. (1998). Nuclear access and action of Notch in vivo. Cell 93,649 -660.[Medline]
Struhl, G., Fitzgerald, K. and Greenwald, I. (1993). Intrinsic activity of the Lin-12 and Notch intracellular domains in vivo. Cell 74,331 -345.[Medline]
Struhl, G. and Greenwald, I. (1999). Presenilin is required for activity and nuclear access of Notch in Drosophila.Nature 398,522 -525.[CrossRef][Medline]
Thomas, U., Speicher, S. A. and Knust, E. (1991). The Drosophila gene Serrate encodes an EGF-like transmembrane protein with a complex expression pattern in embryos and wing discs. Development 111,749 -761.[Abstract]
Wang, Y. and Spellman, M. W. (1998).
Purification and characterization of a GDP-fucose:polypeptide
fucosyltransferase from Chinese hamster ovary cells. J. Biol.
Chem. 273,8112
-8118.
Wang, Y., Shao, L., Shi, S., Harris, R. J., Spellman, M. W.,
Stanley, P. and Haltiwanger, R. S. (2001). Modification of
epidermal growth factor-like repeats with O-fucose. Molecular cloning
and expression of a novel GDP-fucose protein O-fucosyltransferase.
J. Biol. Chem. 276,40338
-40345.
Wharton, K. A., Johansen, K. M., Xu, T. and Artavanis-Tsakonas, S. (1985). Nucleotide sequence from the neurogenic locus Notch implies a gene product that shares homology with proteins containing EGF-like repeats. Cell 43,567 -581.[Medline]
Wiggins, C. A. and Munro, S. (1998). Activity
of the yeast MNN1 -1, 3-mannosyltransferase requires a motif
conserved in many other families of glycosyltransferases. Proc.
Natl. Acad. Sci. USA 95,7945
-7950.
Williams, J. A., Paddock, S. W. and Carroll, S. B.
(1993). Pattern formation in a secondary field: a hierarchy of
regulatory genes subdivides the developing Drosophila wing disc into
discrete subregions. Development
117,571
-584.
Wu, L., Aster, J. C., Blacklow, S. C., Lake, R., Artavanis-Tsakonas, S. and Griffin, J. D. (2000). MAML1, a human homologue of Drosophila mastermind, is a transcriptional co-activator for NOTCH receptors. Nat. Genet. 26,484 -489.[CrossRef][Medline]
Xu, T. and Rubin, G. M. (1993). Analysis of
genetic mosaics in developing and adult Drosophila tissues.
Development 117,1223
-1237.
Yan, Y. T., Liu, J. J., Luo, Y., Chaosu, E., Haltiwanger, R. S.,
Abate-Shen, C. and Shen, M. M. (2002). Dual roles of Cripto
as a ligand and coreceptor in the nodal signaling pathway. Mol.
Cell. Biol. 22,4439
-4449.
Ye, Y., Lukinova, N. and Fortini, M. E. (1999). Neurogenic phenotypes and altered Notch processing in Drosophila Presenilin mutants. Nature 398,525 -529.[CrossRef][Medline]
Zeng, C., Younger-Shepherd, S., Jan, L. Y. and Jan, Y. N.
(1998). Delta and Serrate are redundant Notch ligands required
for asymmetric cell divisions within the Drosophila sensory organ
lineage. Genes Dev. 12,1086
-1091.
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