1 Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK
2 School of Biological Sciences, University of Manchester, 3.239 Stopford
Building, Oxford Road, Manchester M13 9PT, UK
3 Harvard Medical School/HHMI, Department of Genetics, 77 Avenue Louis Pasteur,
NRB#339, Boston, MA 02115, USA
* Author for correspondence (e-mail: ama11{at}cus.cam.ac.uk)
Accepted 2 February 2005
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SUMMARY |
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Key words: Notch, Armadillo, Signalling, Wnt
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Introduction |
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There is evidence that Notch can also signal in a Su(H)-independent manner
(Endo et al., 2002;
Endo et al., 2003
;
Martinez Arias et al., 2002
).
A number of experiments in Drosophila indicate that this alternative
pathway modulates signalling by Wingless, a member of the Wnt family of
signalling molecules (Martinez Arias et
al., 2002
). Loss of function of Notch, but not of
Delta or of Su(H), can bypass loss of function of
wingless, or of dishevelled, a gene that encodes a core
element in the transduction of the Wnt signal
(Brennan et al., 1999a
;
Lawrence et al., 2001
). This
suggests that Notch can downregulate Wnt signalling in a Su(H)-independent
manner, a notion reinforced by the existence of gain-of-function mutations in
Notch, which antagonise Wingless signalling
(Brennan et al., 1999b
;
Martinez Arias et al., 2002
;
Ramain et al., 2001
).
Consistent with these observations, removal of Notch1 in the skin
leads to tumours associated with Wnt signalling and with high levels of the
nuclear form of ß-catenin (Nicolas et
al., 2003
). However, even though the interaction between Notch and
Wingless signalling is well established at the genetic level its molecular
mechanism remains unclear.
It is generally accepted that the key parameter of Wnt signalling is the
stability and precise intracellular location of a soluble pool of
Armadillo/ß-catenin (Arm/ß-cat)
(Gottardi and Gumbiner, 2001).
In the absence of Wnt this pool interacts with a destruction complex where it
is phosphorylated by Shaggy/GSK3ß and degraded via the proteasome. Wnt
acting through the Frizzled and Arrow/LRP receptors activates the cytoplasmic
adaptor protein Dishevelled which, in a poorly understood manner leads to the
inactivation of the destruction complex and allows the accumulation of a
hypophosphorylated form of Armadillo/ß-catenin. This form then enters the
nucleus where it interacts with members of the TCF family of transcription
factors to influence the transcriptional state of the cell
(Tolwinski and Wieschaus,
2004a
). While the central role of Armadillo/ß-catenin is well
established, the mechanism by which it is activated remains open to discussion
(Giles et al., 2003
;
Tolwinski and Wieschaus,
2004b
). It has recently been observed that Axin has effects on Wnt
signalling that are independent of Shaggy/GSK3ß
(Tolwinski et al., 2003
),
suggesting that the central event in the activation of
Armadillo/ß-catenin is the activity of Axin.
Here we analyse the molecular nature of the interactions between Notch and Wingless signalling in Drosophila and between mouse Notch1 and ß-catenin.
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Materials and methods |
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Imaginal wing disc were dissected from third instar larvae in fix solution [4% paraformaldehyde in balanced salt solution (BBS) with 1 mM CaCl2]. Discs were fixed for 30 minutes and then immunostained with the indicated antibodies in BBS [50 mM BES, 280 mM NaCl, 1.5 mM Na2HPO4.2H2O]+ 0.1% Triton X-100, 0.5% BSA 1 mM CaCl2] using standard antibody staining protocols. Discs were mounted in Vectashield and viewed using a confocal microscope (note, the same gain was used in each figure set).
Analysis of Armadillo protein levels
Wing discs from third instar larvae expressing Armadillos10,
TNotch, FLNotch or NICD under the control of dppGAL4 were lysed in
2xLaemmli buffer (Harlow and Lane,
1988). Proteins were separated by 8% SDS-PAGE, the equivalent of
five wing discs were loaded per lane. Western blot analysis for Armadillo (N2
7A1), Armadillos10 (anti-MYC, 9E10) and tubulin (E7) was
performed.
Cell based reporter assays
Assays in insect cells
Transfections were performed in triplicate in 96-well plates using
8x104 cells per well and Effectene transfection reagent
(Qiagen). The amount of Firefly and Renilla luciferase was measured 4 days
after transfection using Dual-Glo reagent (Promega). Data is normalised with
respect to Renilla luciferase and presented as relative light units (RLU), all
data represents at least three independent experiments.
RNAi experiment
Clone 8 cells (Peel et al.,
1990) were used. dsRNAs were synthesised as described previously
(Boutros et al., 2004
); 80 ng
of dsRNA was added to each transfection reaction along with luciferase
reporter (Top12X-HS-luciferase; R.G. and N.P., unpublished data), and
normalisation vector (pPOLIII-Renilla) in a 1:1 ratio, 50 ng of total DNA
added per well.
Gain of function assays
SL2 (Nagao et al., 1996)
and S2R+ (Yanagawa et al.,
1998
) cells were used. For each transfection the ratio of
luciferase reporter (Top12X-HS-luciferase), normalisation vector
(pPOLIII-Renilla) and inducer [pPac-S37Aßcat
(Schweizer and Varmus, 2003
)]
DNA was 1:1:2, the remaining DNA was composed of variable amounts of pPACTN
and pPAC with a total amount 200 ng DNA added per well.
Assays in mammalian cells
Two Notch1 (Nye et al.,
1994) constructs bearing extracellular deletions were generated in
pSecTag2 (Invitrogen): LNR-N1, which lacks amino acids 19-1654, so the encoded
protein should be identical to that produced following furin cleavage at the
S1 site, and
N-N1, which lacks amino acids 19-1710, so the encoded
protein should be identical to that produced upon cleavage at the S2 site
during ligand-induced signalling. Plasmids encoding mouse Wnt1
(Shimizu et al., 1997
),
Xenopus dishevelled (Sokol,
1996
) and Xenopus ß-catenin
(Kypta et al., 1996
) have been
described previously. The Lef1-VP16 fusion protein and the CBF reporter were
obtained from Dr R. Kemler, Max-Planck Institut für Immunbiologie and Dr
G. McKenzie, Lorantis Ltd. Triplicate transfections were performed in 24-well
plates with HEK-293T cells (1x105 cells/well), using the
calcium phosphate co-precipitation method with a plasmid cocktail containing
0.22 µg of DNA (including 50 ng pTOPFLASH or CBF1 luciferase reporter, and
20 ng pRL-CMV). Lysates were prepared 48 hours after transfection, and Firefly
and Renilla luciferase activities in 5 µl of lysate were measured with the
Dual Luciferase Reagent (Promega).
Immunoprecipitation experiments
Wild-type Drosophila embryos were dechorionated and lysed in RIPA
or NP-40 buffer (Harlow and Lane,
1988). Each immunoprecipitation reaction contained the equivalent
of 5 µl packed volume of embryos homogenised in 250 µl lysis buffer.
Notch proteins were immunoprecipitated with 20 µl anti-NICD sheep antiserum
or 50 µl anti-Notch (C17.9C6) and 20 µl protein G Sepharose. Armadillo
proteins were immunoprecipitated using 10 µl anti-Armadillo rabbit
antiserum and 20 µl protein A Sepharose. Control reactions with protein G,
protein A or anti-GFP rabbit antiserum with Protein A were undertaken. Immune
complexes were released by boiling in 60 µl Laemmli buffer and separated by
8% SDS-PAGE, 20 µl immunoprecipitate per lane. Proteins were detected by
western blot.
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Results |
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The inputs of Notch and Wingless signalling on the development of the wing
are well characterised (Klein and Martinez
Arias, 1998; Klein and
Martinez Arias, 1999
; Martinez
Arias, 2003
). Notch and Wingless signalling cooperate in the
development of the wing and in the case of Notch the effects are mediated by
NICD. To test if the cleavage-independent function of Notch modulates Wingless
signalling, we have expressed NICD and TNotch at the same time that we
activate Wingless signalling either with ectopic expression of Wingless or of
a constitutively active form of Armadillo, Armadillos10. This form
of Armadillo lacks the Shaggy/GSK3ß phosphorylation sites and provides
Wingless-independent signalling by escaping degradation by the Axin-based
destruction complex (Pai et al.,
1997
). Expression of either Wingless or Armadillos10
along the AP boundary results in an expansion of the hinge region and the
occasional appearance of extra wing tissue off the notum
(Klein and Martinez Arias,
1998
) (Fig. 1C).
However, the effects of the intracellular domain of Notch depend on its
molecular disposition. Expression of NICD along the AP boundary induces the
appearance of an ectopic wing margin and promotes the growth of the wing
(Diaz-Benjumea and Cohen,
1995
; Klein and Martinez
Arias, 1998
), while expression of TNotch leads to a slight
reduction in the overall size of the wing pouch region of the disc
(Fig. 1B). In the developing
wing, co-expression of NICD with either Wingless or Armadillos10
leads to a synergistic effect of extra growth of the wing tissue
(Klein and Martinez Arias,
1998
). In contrast to NICD, TNotch is very effective in
suppressing the effects of ectopic expression of Wingless and, surprisingly,
also of Armadillos10 (Fig.
1D, also see Fig. S1 in supplementary material).
|
|
Altogether these observations suggest that there is an activity of Notch, independent of Su(H), which modulates the Wingless signalling pathway at or below the level of Armadillo.
Torso-Notch modulates the levels of Armadillo
The effects of Notch on the activity of Armadillos10 could be
due to a squelching of GAL4 by the UASTNotch construct reducing the expression
of other constructs co-expressed with it. However in situ experiments
demonstrate that UASTNotch transcription does not affect
UASArmadillos10 expression (P.H., unpublished). This suggests that
the effects of Notch on the activity of Armadillo result either from a
parallel input on Wingless target gene expression or from an effect on
Armadillo itself. In order to test this we have analysed the effects of Notch
on the levels, state and localisation of the Armadillo protein.
In the epithelium of the wing disc, Armadillo is preferentially localised
at the level of the adherens junctions
(Fig. 1E,F) and exists in at
least two different phosphorylation states
(Fig. 3F) that have been
correlated with function (Peifer et al.,
1994a): a hypophosphorylated form that has been associated with
nuclear activity (Staal et al.,
2002
) and a hyperphosphorylated form that is predominantly
restricted to the adherens junctions
(Peifer et al., 1994b
). In our
experiments, when Armadillos10 is expressed it becomes
preferentially localised to the adherens junctions
(Fig. 1E). The expression of
Armadillos10 has a significant effect on the endogenous Armadillo,
which is displaced from the adherens junctions and accumulates in the
cytoplasm (Fig. 1C,F). In
western blots this is translated into a rise in endogenous Armadillo levels
and is correlated with an increase in the proportion of the hypophosphorylated
form (Fig. 3E,F). These effects
are likely to be associated with the enhanced stability of
Armadillos10 and its ability to interact with and titrate the
activity of components of the Armadillo destruction complex
(Cox et al., 1999
;
Pai et al., 1997
).
|
To test further the effects of Notch on Armadillo we overexpressed full
length Armadillo together with TNotch. When Armadillo is overexpressed on its
own, it accumulates to very high levels in the cytoplasm of the cells
(Marygold and Vincent, 2003)
in a manner that is strictly dependent on Wingless signalling and other less
characterised factors (see Fig. S3 in supplementary material). This
accumulation is significantly reduced in the presence of TNotch
(Fig. 3C,D).
This data demonstrates that in the imaginal wing disc the activity and levels of both Armadillo and Armadillos10, a form that mimics oncogenic forms of ß-catenin, are the subject of regulation by the Notch receptor.
Notch modulates the transcriptional activity of Armadillo
These results demonstrate a regulatory effect of Notch on the concentration
and transcriptional effects of an activated form of Armadillo in vivo.
Although our observations suggest a direct effect of Notch on the activity of
Armadillo, the complexity of the in vivo regulatory networks could conceivably
create situations that would produce the observed effects indirectly. To rule
this out, and analyse the interaction further, we studied the effects of Notch
loss and gain of function on Wnt signalling in Drosophila cells in
culture by measuring the effects of Notch on the activity of a Wnt reporter
(TOP12).
If gain of function of Notch suppresses Wnt signalling in the wing disc, we
asked what would happen to Wnt signalling in the absence of Notch. Earlier
experiments in vivo have shown that removal of Notch results in ectopic
activity of a Wnt reporter (Lawrence et
al., 2001). We tested this in culture using Clone8 cells (cl8), a
diploid cell population derived from wing imaginal discs that have been used
for a variety of assays of Wnt activity
(van Leeuwen et al., 1994
).
The TOP12 reporter is functional in these cells and is activated by Wnt
signalling in a dose-dependent manner (R.G., unpublished data). Strong
activation of the reporter is also observed in these cells when Notch
signalling is reduced by targeted RNA interference (RNAi) of the
Notch gene (Fig. 4B). In these experiments, four different dsRNA molecules directed against the
coding region of the intracellular domain of Notch resulted in a
quantitatively different but qualitatively similar effect (P.H., unpublished
data). These results confirm that Notch exerts a negative effect on Wnt
signalling.
|
These results confirm and extend our observations in vivo and support the
notion that the effects of Notch on Wnt signalling are mediated through a
direct negative regulation of the activity of Armadillo. To test whether these
effects are restricted to Drosophila Notch we have tested the ability
of mouse Notch1 to modulate Wnt signalling in HEK-293T cells. A previous
report has indicated that Notch1 NICD can suppress ß-catenin-mediated Wnt
signalling in Notch1 mutant keratinocytes
(Nicolas et al., 2003). We
have tested the ability of two different forms of membrane tethered Notch1 to
modulate Wnt signalling (Fig.
4E,F). One form
NN1, a version of
E that
removes all but 13 amino acids of the extracellular domain
(Mumm et al., 2000
)
(Fig. 4A), can undergo
spontaneous cleavage and activate a CBF reporter
(Fig. 4H). This form can also
suppress ß-catenin activity (Fig.
4E). A second membrane-tethered form LNR-N1
(Fig. 4A), a version of
NLNR is rarely cleaved (Mumm et
al., 2000
) and only activates the CBF reporter very weakly
(Fig. 4H), but still strongly
suppresses the activity of ß-catenin
(Fig. 4F).
These observations, together with the observation that Notch cannot inhibit
Wnt reporter activity driven by a LEF1-VP16 fusion protein confirm and extend
the results from Drosophila that indicate that Notch has an ability
to interfere with the activity of ß-catenin. They also support the notion
that this effect might not require the cleavage of Notch or its ability to
activate transcription. The effects of Notch on the activity of ß-catenin
contrast with those of a soluble form of Frizzled8 (ExFz8) which, as shown
previously are effective in suppressing Wnt-induced Wnt signalling
(Brennan et al., 2004) but are
not able to suppress ß-catenin-induced Wnt signalling
(Fig. 4G).
Notch can regulate Armadillo independently of Sgg/GSK3ß
The results described above show that Notch modulates the amount and the
activity of Armadillo and that this effect is different from that mediated by
NICD. To explore these relationships further we have analysed the effects of
loss of Notch function on the stability of Armadillo.
In the imaginal discs, cells lacking shaggy function exhibit elevated levels of Armadillo that is enriched in the neighbourhood of the adherens junctions (Fig. 5A-C). In contrast, loss of Notch function does not alter the levels of endogenous Armadillo in a reproducible manner, although some times we have observed an increased accumulation of Armadillo in the neighbourhood of the DV boundary (see legend to Fig. 5). However, simultaneous loss of shaggy and Notch function results in small clones of cells in which Armadillo is not restricted to the adherens junctions as it is in shaggy mutants, but it is now distributed throughout the cytoplasm (Fig. 5D-F).
|
The relationships that we have observed between Notch and Armadillo, as well as between Notch and a form of Armadillo that is resistant to Shaggy-mediated degradation, led us to enquire whether Notch could reverse the effects of removal of Shaggy/GSK3ß. To do this we expressed TNotch in clones of cells that had lost shaggy in the developing wing disc. Loss of shaggy generates large clones with cell autonomous high levels of Wnt signalling, as revealed by high levels of Armadillo and ectopic expression of targets of Wingless signalling, e.g. senseless (Fig. 6A-D). When TNotch is expressed in cells that have lost shaggy, Armadillo is returned to wild-type levels and the transcriptional response is abolished (Fig. 6E-H).
|
Armadillo associates with Notch in Drosophila
Our observations indicate a close functional association between Armadillo
and Notch. One possibility is that the effects of Notch are indirect and are
mediated by some proteins associated with a Su(H)-independent activity.
Although this may be the case, it is also possible that Armadillo is part of
this complex. This possibility is suggested by the observation that Armadillo
and Notch show a high degree of co-localisation at the adherens junction of
the epidermal cells of the wing disc
(Fehon et al., 1991;
Lamb et al., 1998
). To test
whether Notch and Armadillo are associated in the cell, we immunoprecipitated
Notch from developing embryos and searched for Armadillo amongst the
co-immunoprecipitated proteins. Two different anti-Notch antibodies were used
and in both cases Armadillo protein was detected in the same protein complex
as the immunoprecipitated Notch protein
(Fig. 7). Interestingly, the
predominant form of Notch protein detected in these assays is unprocessed and
uncleaved (Kidd and Lieber,
2002
), suggesting that this complex is membrane associated. The
reverse experiment, in which Armadillo protein is immunoprecipitated, was also
undertaken; here an unprocessed and uncleaved form of Notch was found to be
associated with Armadillo (P.H., unpublished data). Previous experiments have
indicated that Dishevelled, another element of Wnt signalling, can associate
with Notch in a yeast two-hybrid assay. We have confirmed this and further
shown this association in the same immunoprecipitates from embryos in which we
find the complex between Notch and Armadillo
(Fig. 7). Other proteins such
as E-cadherin and nuclear lamin were not detected in the immunoprecipitates
(see Fig. S5 in supplementary material).
|
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Discussion |
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Here we have shown that Notch signalling provides an important input into Wnt signalling in Drosophila by associating with Armadillo and regulating its levels and activity during Wingless signalling (Fig. 8). This activity of Notch, which is different and probably independent of that which mediates CBF1/Su(H)-dependent signalling, lies functionally downstream of Shaggy/GSK3ß and targets the concentration and activity of the hypophosphorylated form of Armadillo. It can also modulate the activity of an oncogenic form of vertebrate ß-catenin and we have demonstrated that this functional interaction between Notch and Armadillo extends to the vertebrate system, with mNotch1 regulating the activity of ß-catenin in tissue culture cells.
|
Previous studies have implicated Deltex and Dishevelled as important
elements of the interaction between Notch and Wingless signalling
(Axelrod et al., 1996;
Martinez Arias et al., 2002
;
Ramain et al., 2001
). Both
proteins bind Notch, but they do so in different places. Deltex binds to the
cdc10/ANK repeats (Matsuno et al.,
1995
) and promotes Su(H)-independent Notch signalling. Whereas,
Dishevelled binds within a broad region C-terminal to this domain and reduces
the Su(H)-independent activity of Notch
(Axelrod et al., 1996
;
Ramain et al., 2001
;
Zecchini et al., 1999
). Here
we have shown that Armadillo also interacts with Notch, probably through the
same broad region that binds Dishevelled. Mutations in Notch that impair this
domain result in Notch receptors that interfere with Wnt signalling
(Ramain et al., 2001
) and we
have observed that its deletion reduces the efficiency with which the
intracellular domain of Notch affects the levels and activity of Armadillo
(P.H., unpublished data). Together these observations underscore the role of
this region of Notch in mediating interactions between Notch and Wnt
signalling by targeting the active form of Armadillo/ß-catenin.
The relationship between Notch and Armadillo in Drosophila extends
to their vertebrate homologues, Notch1 and ß-catenin. This interaction,
rather than an interaction of Dishevelled with Notch/CBF signalling, might
reflect the functional relationships between the two signalling systems that
have been reported during the development of the skin
(Lowell et al., 2000;
Nicolas et al., 2003
;
Zhu and Watt, 1999
), the
immune system (Radtke et al.,
1999
; Reya et al.,
2000
) and in somitogenesis
(Aulehla et al., 2003
;
Dale et al., 2003
;
Pourquie, 2003
). In these
instances Wnt and Notch drive alternative fates (skin and immune system) or
act antagonistically (somites) perhaps by a combination of their individual
pathways and the modulatory interaction that we have described here. One
consequence of this modulatory interaction might also be the observed tumour
suppressor function of Notch1 in the mouse skin where removal of
Notch1 results in the generation of tumours associated with an
increase in the levels of active ß-catenin and Wnt signalling
(Nicolas et al., 2003
). Whilst
some of the elevation of ß-catenin in these cells might be a secondary
consequence of activation of Wnt signalling, our observations suggests that
the loss of Notch1 can also contribute to this increase by allowing
the activation of ß-catenin. In a different study carboxyl-terminal
deletions in Notch1, which include the region that binds Dishevelled
and Armadillo, enhanced the oncogenic effects of a chimeric E2A-PBX1 protein
(Feldman et al., 2000
). It is
possible that some of this effect is due to misregulation of ß-catenin in
the tumours.
In summary, we have shown that Notch provides a modulatory input in the
activity of Armadillo/ß-catenin (Fig.
8). This modulation provides two functions: it establishes a
threshold for Wnt signalling that is likely to play an important role in the
patterning of tissues and the assignation of cell fates during development
(Martinez Arias, 2002) and, in
addition it provides a stringent regulation of the activated form of
Armadillo/ß-catenin. The second function might be crucial in pathological
situations and might contribute to the understanding of Notch as a tumour
suppressor (Radtke and Raj,
2003
).
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/8/1819/DC1
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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.
Aulehla, A., Wehrle, C., Brand-Saberi, B., Kemler, R., Gossler, A., Kanzler, B. and Herrmann, B. G. (2003). Wnt3a plays a major role in the segmentation clock controlling somitogenesis. Dev. Cell 4,395 -406.[CrossRef][Medline]
Axelrod, J. D., Matsuno, K., Artavanis-Tsakonas, S. and Perrimon, N. (1996). Interaction between Wingless and Notch signaling pathways mediated by dishevelled. Science 271,1826 -1832.[Abstract]
Barolo, S., Stone, T., Bang, A. G. and Posakony, J. W.
(2002). Default repression and Notch signaling: Hairless acts as
an adaptor to recruit the corepressors Groucho and dCtBP to Suppressor of
Hairless. Genes Dev. 16,1964
-1976.
Boutros, M., Kiger, A. A., Armknecht, S., Kerr, K., Hild, M.,
Koch, B., Haas, S. A., Consortium, H. F., Paro, R. and Perrimon, N.
(2004). Genome-wide RNAi analysis of growth and viability in
Drosophila cells. Science
303,832
-835.
Brennan, K., Tateson, R., Lewis, K. and Martinez Arias, A.
(1997). A functional analysis of Notch mutations in Drosophila.
Genetics 147,177
-188.
Brennan, K., Klein, T., Wilder, E. and Martinez Arias, A. (1999a). Wingless modulates the effects of dominant negative notch molecules in the developing wing of Drosophila. Dev. Biol. 216,210 -229.[CrossRef][Medline]
Brennan, K., Tateson, R., Lieber, T., Couso, J. P., Zecchini, V. and Matinez Arias, A. (1999b). The abruptex mutations of notch disrupt the establishment of proneural clusters in Drosophila. Dev. Biol. 216,230 -242.[CrossRef][Medline]
Brennan, K., Gonzalez-Sancho, J. M., Castelo-Soccio, L. A., Howe, L. R. and Brown, A. M. (2004). Truncated mutants of the putative Wnt receptor LRP6/Arrow can stabilize beta-catenin independently of Frizzled proteins. Oncogene 23,4873 -4884.[CrossRef][Medline]
Couso, J. P., Bate, M. and Martinez-Arias, A. (1993). A wingless-dependent polar coordinate system in Drosophila imaginal discs. Science 259,484 -489.[Medline]
Cox, R. T., Pai, L. M., Miller, J. R., Orsulic, S., Stein, J.,
McCormick, C. A., Audeh, Y., Wang, W., Moon, R. T. and Peifer, M.
(1999). Membrane-tethered Drosophila Armadillo cannot
transduce Wingless signal on its own. Development
126,1327
-1335.
Dale, J. K., Maroto, M., Dequeant, M. L., Malapert, P., McGrew, M. and Pourquie, O. (2003). Periodic notch inhibition by lunatic fringe underlies the chick segmentation clock. Nature 421,275 -278.[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]
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.
Endo, Y., Osumi, N. and Wakamatsu, Y. (2002). Bimodal functions of Notch-mediated signaling are involved in neural crest formation during avian ectoderm development. Development 129,863 -873.[Medline]
Endo, Y., Osumi, N. and Wakamatsu, Y. (2003). Deltex/Dtx mediates NOTCH signaling in regulation of Bmp4 expression in cranial neural crest formation during avian development. Dev. Growth Differ. 45,241 -248.[CrossRef][Medline]
Fehon, R. G., Johansen, K., Rebay, I. and Artavanis-Tsakonas, S. (1991). Complex cellular and subcellular regulation of notch expression during embryonic and imaginal development of Drosophila: implications for notch function. J. Cell Biol. 113,657 -669.[Abstract]
Feldman, B. J., Hampton, T. and Cleary, M. L.
(2000). A carboxy-terminal deletion mutant of Notch1 accelerates
lymphoid oncogenesis in E2A-PBX1 transgenic mice.
Blood 96,1906
-1913.
Furriols, M. and Bray, S. (2001). A model Notch response element detects Suppressor of Hairless-dependent molecular switch. Curr. Biol. 11,60 -64.[CrossRef][Medline]
Giles, R. H., van Es, J. H. and Clevers, H. (2003). Caught up in a Wnt storm: Wnt signaling in cancer. Biochim. Biophys. Acta 1653, 1-24.[Medline]
Gottardi, C. J. and Gumbiner, B. M. (2001). Adhesion signaling: how beta-catenin interacts with its partners. Curr. Biol. 11,R792 -R794.[CrossRef][Medline]
Guger, K. A. and Gumbiner, B. M. (2000). A mode of regulation of beta-catenin signaling activity in Xenopus embryos independent of its levels. Dev. Biol. 223,441 -448.[CrossRef][Medline]
Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Kidd, S. and Lieber, T. (2002). Furin cleavage is not a requirement for Drosophila Notch function. Mech. Dev. 115,41 -51.[CrossRef][Medline]
Kidd, S., Lieber, T. and Young, M. W. (1998).
Ligand-induced cleavage and regulation of nuclear entry of Notch in Drosophila
melanogaster embryos. Genes Dev.
12,3728
-3740.
Klein, T. and Martinez Arias, A. (1998). Different spatial and temporal interactions between Notch, wingless, and vestigial specify proximal and distal pattern elements of the wing in Drosophila. Dev. Biol. 194,196 -212.[CrossRef][Medline]
Klein, T. and Martinez Arias, A. (1999). The
vestigial gene product provides a molecular context for the interpretation of
signals during the development of the wing in Drosophila.Development 126,913
-925.
Klein, T., Seugnet, L., Haenlin, M. and Martinez Arias, A.
(2000). Two different activities of Suppressor of Hairless during
wing development in Drosophila. Development
127,3553
-3566.
Kopan, R. (2002). Notch: a membrane-bound
transcription factor. J. Cell Sci.
115,1095
-1097.
Kopan, R., Nye, J. S. and Weintraub, H. (1994).
The intracellular domain of mouse Notch: a constitutively activated repressor
of myogenesis directed at the basic helix-loop-helix region of MyoD.
Development 120,2385
-2396.
Kypta, R. M., Su, H. and Reichardt, L. F. (1996). Association between a transmembrane protein tyrosine phosphatase and the cadherin-catenin complex. J. Cell Biol. 134,1519 -1529.[Abstract]
Lamb, R. S., Ward, R. E., Schweizer, L. and Fehon, R. G.
(1998). Drosophila coracle, a member of the protein 4.1
superfamily, has essential structural functions in the septate junctions and
developmental functions in embryonic and adult epithelial cells.
Mol. Biol. Cell 9,3505
-3519.
Lawrence, N., Klein, T., Brennan, K. and Martinez Arias, A.
(2000). Structural requirements for Notch signalling with Delta
and Serrate during the development and patterning of the wing disc of
Drosophila. Development
127,3185
-3195.
Lawrence, N., Langdon, T., Brennan, K. and Martinez Arias, A. (2001). Notch signaling targets the Wingless responsiveness of a Ubx visceral mesoderm enhancer in Drosophila. Curr. Biol. 11,375 -385.[CrossRef][Medline]
Lee, E., Salic, A. and Kirschner, M. W. (2001).
Physiological regulation of [beta]-catenin stability by Tcf3 and CK1epsilon.
J. Cell Biol. 154,983
-993.
Lee, E., Salic, A., Kruger, R., Heinrich, R. and Kirschner, M. W. (2003). The roles of apc and axin derived from experimental and theoretical analysis of the Wnt pathway. PLoS Biol. 1,E10 .[Medline]
Lieber, T., Kidd, S., Alcamo, E., Corbin, V. and Young, M. W. (1993). Antineurogenic phenotypes induced by truncated Notch proteins indicate a role in signal transduction and may point to a novel function for Notch in nuclei. Genes Dev. 7,1949 -1965.[Abstract]
Lowell, S., Jones, P., le Roux, I., Dunne, J. and Watt, F. M. (2000). Stimulation of human epidermal differentiation by delta-notch signalling at the boundaries of stem-cell clusters. Curr. Biol. 10,491 -500.[CrossRef][Medline]
Martinez Arias, A. (2002). New alleles of Notch draw a blueprint for multifunctionality. Trends Genet. 18,168 -170.[CrossRef][Medline]
Martinez Arias, A. (2003). Wnts as morphogens? The view from the wing of Drosophila. Nat. Rev. Mol. Cell Biol. 4,321 -325.[CrossRef][Medline]
Martinez Arias, A., Zecchini, V. and Brennan, K. (2002). CSL-independent Notch signalling: a checkpoint in cell fate decisions during development? Curr. Opin. Genet. Dev. 12,524 .[CrossRef][Medline]
Marygold, S. J. and Vincent, J. P. (2003). Armadillo levels are reduced during mitosis in Drosophila. Mech. Dev. 120,157 -165.[CrossRef][Medline]
Matsuno, K., Diederich, R. J., Go, M. J., Blaumueller, C. M. and
Artavanis-Tsakonas, S. (1995). Deltex acts as a positive
regulator of Notch signaling through interactions with the Notch ankyrin
repeats. Development
121,2633
-2644.
Mumm, J. S., Schroeter, E. H., Saxena, M. T., Griesemer, A., Tian, X., Pan, D. J., Ray, W. J. and Kopan, R. (2000). A ligand-induced extracellular cleavage regulates gamma-secretase-like proteolytic activation of Notch1. Mol. Cell 5, 197-206.[CrossRef][Medline]
Nagao, M., Ebert, B. L., Ratcliffe, P. J. and Pugh, C. W. (1996). Drosophila melanogaster SL2 cells contain a hypoxically inducible DNA binding complex which recognises mammalian HIF-binding sites. FEBS Lett 387,161 -166.[CrossRef][Medline]
Nicolas, M., Wolfer, A., Raj, K., Kummer, J. A., Mill, P., van Noort, M., Hui, C. C., Clevers, H., Dotto, G. P. and Radtke, F. (2003). Notch1 functions as a tumor suppressor in mouse skin. Nat. Genet. 33,416 -421.[CrossRef][Medline]
Nye, J. S., Kopan, R. and Axel, R. (1994). An
activated Notch suppresses neurogenesis and myogenesis but not gliogenesis in
mammalian cells. Development
120,2421
-2430.
Pai, L. M., Orsulic, S., Bejsovec, A. and Peifer, M.
(1997). Negative regulation of Armadillo, a Wingless effector in
Drosophila. Development
124,2255
-2266.
Peel, D. J., Johnson, S. A. and Milner, M. J. (1990). The ultrastructure of imaginal disc cells in primary cultures and during cell aggregation in continuous cell lines. Tissue Cell 22,749 -758.[Medline]
Peifer, M., Pai, L. M. and Casey, M. (1994a). Phosphorylation of the Drosophila adherens junction protein Armadillo: roles for wingless signal and zeste-white 3 kinase. Dev. Biol. 166,543 -556.[CrossRef][Medline]
Peifer, M., Sweeton, D., Casey, M. and Wieschaus, E.
(1994b). Wingless signal and Zeste-white 3 kinase trigger
opposing changes in the intracellular distribution of Armadillo.
Development 120,369
-380.
Polakis, P. (2000). Wnt signaling and cancer.
Genes Dev. 14,1837
-1851.
Pourquie, O. (2003). The segmentation clock:
converting embryonic time into spatial pattern.
Science 301,328
-330.
Radtke, F. and Raj, K. (2003). The role of Notch in tumorigenesis: oncogene or tumour suppressor? Nat. Rev. Cancer 3,756 -767.[CrossRef][Medline]
Radtke, F., Wilson, A., Stark, G., Bauer, M., van Meerwijk, J., MacDonald, H. R. and Aguet, M. (1999). Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10,547 -558.[CrossRef][Medline]
Ramain, P., Khechumian, K., Seugnet, L., Arbogast, N., Ackermann, C. and Heitzler, P. (2001). Novel Notch alleles reveal a Deltex-dependent pathway repressing neural fate. Curr. Biol. 11,1729 -1738.[CrossRef][Medline]
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.[CrossRef][Medline]
Rebay, I., Fehon, R. G. and Artavanis-Tsakonas, S. (1993). Specific truncations of Drosophila Notch define dominant activated and dominant negative forms of the receptor. Cell 74,319 -329.[CrossRef][Medline]
Reya, T., O'Riordan, M., Okamura, R., Devaney, E., Willert, K., Nusse, R. and Grosschedl, R. (2000). Wnt signaling regulates B lymphocyte proliferation through a LEF-1 dependent mechanism. Immunity 13,15 -24.[CrossRef][Medline]
Schroeter, E. H., Kisslinger, J. A. and Kopan, R. (1998). Notch-1 signalling requires ligand-induced proteolytic release of intracellular domain. Nature 393,382 -386.[CrossRef][Medline]
Schweisguth, F. (2004). Notch signaling activity. Curr. Biol. 14,R129 -R138.[CrossRef][Medline]
Schweisguth, F. and Lecourtois, M. (1998). The activity of Drosophila Hairless is required in pupae but not in embryos to inhibit Notch signal transduction. Dev. Genes Evol. 208, 19-27.[CrossRef][Medline]
Schweizer, L. and Varmus, H. (2003). Wnt/Wingless signaling through beta-catenin requires the function of both LRP/Arrow and frizzled classes of receptors. BMC Cell Biol. 4,4 .[CrossRef][Medline]
Seugnet, L., Simpson, P. and Haenlin, M. (1997). Requirement for dynamin during Notch signaling in Drosophila neurogenesis. Dev. Biol. 192,585 -598.[CrossRef][Medline]
Shimizu, H., Julius, M. A., Giarre, M., Zheng, Z., Brown, A. M. and Kitajewski, J. (1997). Transformation by Wnt family proteins correlates with regulation of beta-catenin. Cell Growth Differ. 8,1349 -1358.[Abstract]
Sokol, S. Y. (1996). Analysis of Dishevelled signalling pathways during Xenopus development. Curr. Biol. 6,1456 -1467.[CrossRef][Medline]
Staal, F. J., Noort Mv, M., Strous, G. J. and Clevers, H. C.
(2002). Wnt signals are transmitted through N-terminally
dephosphorylated beta-catenin. EMBO Rep.
3, 63-68.
Struhl, G. and Adachi, A. (1998). Nuclear access and action of notch in vivo. Cell 93,649 -660.[CrossRef][Medline]
Struhl, G. and Adachi, A. (2000). Requirements for presenilin-dependent cleavage of notch and other transmembrane proteins. Mol. Cell 6,625 -636.[CrossRef][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.[CrossRef][Medline]
Suzuki, H., Watkins, D. N., Jair, K. W., Schuebel, K. E., Markowitz, S. D., Dong Chen, W., Pretlow, T. P., Yang, B., Akiyama, Y., van Engeland, M. et al. (2004). Epigenetic inactivation of SFRP genes allows constitutive WNT signaling in colorectal cancer. Nat. Genet. 36,417 -422.[CrossRef][Medline]
Tolwinski, N. S. and Wieschaus, E. (2001).
Armadillo nuclear import is regulated by cytoplasmic anchor Axin and nuclear
anchor dTCF/Pan. Development
128,2107
-2117.
Tolwinski, N. S. and Wieschaus, E. (2004a). A nuclear function for Armadillo/beta-Catenin. PLoS Biol. 2,E95 .[Medline]
Tolwinski, N. S. and Wieschaus, E. (2004b). Rethinking WNT signaling. Trends Genet. 20,177 -181.[CrossRef][Medline]
Tolwinski, N. S., Wehrli, M., Rives, A., Erdeniz, N., DiNardo, S. and Wieschaus, E. (2003). Wg/Wnt signal can be transmitted through arrow/LRP5,6 and Axin independently of Zw3/Gsk3beta activity. Dev. Cell 4,407 -418.[CrossRef][Medline]
van Leeuwen, F., Samos, C. H. and Nusse, R. (1994). Biological activity of soluble wingless protein in cultured Drosophila imaginal disc cells. Nature 368,342 -344.[CrossRef][Medline]
Wodarz, A. and Nusse, R. (1998). Mechanisms of Wnt signaling in development. Annu. Rev. Cell Dev. Biol. 14,59 -88.[CrossRef][Medline]
Yanagawa, S., Lee, J. S. and Ishimoto, A.
(1998). Identification and characterization of a novel line of
Drosophila Schneider S2 cells that respond to wingless signaling.
J. Biol. Chem. 273,32353
-32359.
Zecca, M., Basler, K. and Struhl, G. (1996). Direct and long-range action of a wingless morphogen gradient. Cell 87,833 -844.[CrossRef][Medline]
Zecchini, V., Brennan, K. and Martinez-Arias, A. (1999). An activity of Notch regulates JNK signalling and affects dorsal closure in Drosophila. Curr. Biol. 9, 460-469.[CrossRef][Medline]
Zhu, A. J. and Watt, F. M. (1999). Beta-catenin
signalling modulates proliferative potential of human epidermal keratinocytes
independently of intercellular adhesion. Development
126,2285
-2298.
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