Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 76100, Israel
* Author for correspondence (e-mail: benny.shilo{at}weizmann.ac.il)
Accepted 26 October 2004
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SUMMARY |
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Key words: Notch, Delta, Serrate, ADAM metalloproteases, Drosophila, Wing development, Kuzbanian-like
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Introduction |
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A sharp distinction between the sending and receiving cells is essential,
because signaling is carried out only between neighboring cells. In some cases
the Notch pathway itself provides the means for initiating the primary
distinction between cells, in a seemingly homogeneous population of cells. It
is thought that random fluctuations elevating the levels of Dl in a given cell
embedded within a group of equivalent cells (e.g. a pro-neural field) will
trigger a further elevation in Dl and a concomitant reduction in Notch levels.
In parallel, activation of Notch in the adjacent (signal-receiving) cells
leads to a reduction in Dl levels
(Heitzler et al., 1996).
In other instances the Notch pathway is dedicated to the refinement of an
already established asymmetry between adjacent cell populations. A case in
point is the definition of vein borders in the pupal wing. The epidermal
growth factor receptor pathway is activated in the future veins, leading to
induction of Dl expression. Localized expression of Dl activates Notch
signaling in the adjacent cells, to inhibit the formation of veins in this
territory (de Celis et al.,
1997; Huppert et al.,
1997
). In this system Notch signaling also relies upon the
simultaneous increase in Dl and decrease of Notch in the sending cells, and
the elimination of Dl in the receiving cells. These responses are at the heart
of maintaining a stable, unidirectional signaling by the Notch pathway.
Which mechanisms contribute to changes in levels of Dl and Notch as a
result of Notch signaling? Transcriptional repression of Dl, mediated by the
Enhancer of split [E(Spl)] complex induced following Notch activation is a
general and direct mechanism contributing to the reduction in Dl levels
(Heitzler et al., 1996;
Hinz et al., 1994
;
Kunisch et al., 1994
). In
other cases, induction of specific transcriptional repressors, such as Cut in
the wing margin, by Notch activation leads to repression of Dl
transcription (de Celis and Bray,
1997
; Micchelli et al.,
1997
).
Mechanisms for Notch protein modification also play a role in maintaining
an asymmetric distribution or activity of Notch. In specific areas, such as
the wing margin, modification of Notch by Fringe, a glycosyltransferase,
renders it refractive to signaling by ligands such as Ser, or conversely more
responsive to Dl expressed by the adjacent cells
(Bruckner et al., 2000;
Okajima and Irvine, 2002
;
Panin et al., 1997
). In
sensory-organ precursor cells, enhanced endocytosis of Notch by asymmetric
segregation of Numb/
-Adaptin was shown to reduce Notch signaling
(Berdnik et al., 2002
;
Frise et al., 1996
). Enhanced
endocytosis of Notch was also observed in Caenorhabditis elegans
vulval development, in the cell that is induced to become the primary source
for the Notch ligand (Shaye and Greenwald,
2002
). In specific areas, such as the wing margin, high levels of
Dl were shown to confer refractivity to Notch signaling, through an
ill-defined dominant-negative effect of Dl
(de Celis and Bray, 1997
;
Micchelli et al., 1997
).
Proteolysis plays an integral part in the Notch signaling pathway,
particularly in sequential cleavages of Notch itself
(Baron, 2003;
Mumm and Kopan, 2000
). The
first cleavage (termed S1), carried out by Furins, bisects Notch at the
extracellular domain, leaving the resulting two polypeptides noncovalently
associated. S1 is a constitutive event. The second cleavage (termed S2) is a
regulated event that takes place only once Notch is bound to Dl presented by
the adjacent cell. This cleavage is thought to be carried out by the Kuzbanian
(Kuz) ADAM metalloprotease (Lieber et al.,
2002
). Initiation of endocytosis of the Dl/Notch complex into the
Dl-presenting cell facilitates this S2 cleavage, and internalizes the
extracellular domain of Notch (Le Borgne
and Schweisguth, 2003
; Parks
et al., 2000
). Finally, the truncated portion of Notch, containing
the transmembrane domain, becomes a target for the Presenilin intra-membrane
protease (S3 cleavage), releasing the cytoplasmic domain of Notch, which is
targeted to the nucleus to induce transcriptional responses
(Struhl and Adachi, 1998
).
Could proteolytic events also contribute to generation or maintenance of
differences in the levels of Dl between sending and receiving cells? The Kuz
metalloprotease was shown to cleave Dl in cell culture and in flies, and to
release a secreted form of Dl
(Mishra-Gorur et al., 2002;
Qi et al., 1999
). However the
role of this cleavage in promoting or suppressing Notch signaling could not be
tested, due to the essential role of Kuz in the regulated S2 cleavage, which
is necessary for activation of Notch
(Lieber et al., 2002
). We
identified an ADAM metalloprotease (termed Kul) dedicated to cleavage of Dl,
and demonstrated the necessity of removing Dl in the cells receiving the
signal, in order to maintain unidirectional Notch signaling.
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Materials and methods |
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To identify flip-out clones in the wing disc expressing only Dl or Dl and Kul, the following cross was carried out. Males carrying UAS-Dl on the X chromosome and UAS-kul/+ on the third chromosome were crossed to females carrying the flipout cassette. Female larvae were selected for dissection. Thus, all larvae expressed Dl in the flipout clones, and half also expressed Kul in the same clones, as identified by Kul antibodies.
Molecular biology
To reconstitute the Kul ORF, RNA from embryos at all stages was isolated by
the Tri reagent (Molecular Research) and was reverse transcribed into a pool
of first strand cDNAs using SuperScript (Invitrogen). This pool served as a
template for PCR, using primers designed according to the BGDP ORF prediction.
The GenBank accession number for the complete ORF (CG1964) is AY525767. For
structure-function studies, different derivatives of Kul protein were made,
using a site-directed mutagenesis kit (Promega). Catalytically inactive Kul
was constructed by generating the E643A mutation in the protease catalytic
site. Kul lacking the intracellular domain (Ex-Kul) was generated by
truncating the protein after residue 991, resulting in a short cytoplasmic
tail of 42 amino acids. The Kul precursor in which pro-domain cleavage was
blocked (Pro-Kul) was made by changing RK at positions 219-220 in the putative
Furin-cleavage site into AG. All Kul constructs (except EX-Kul) were HA tagged
at their C-terminus.
Kul ds-RNA constructs were generated by PCR amplification of antisense 794-1705 followed by sense 340-1705 (where A in the ATG codon is defined as 1). This sequence covers the pro-domain of Kul, and shows no similarity to other Drosophila DNA sequences. The same procedure was carried out with sequences from the Kul cytoplasmic tail (antisense 3381-4425, sense 2911-4425) to form another ds-kul construct.
Full-length DTACE sequence (CG7908, GenBank accession number AY525768) from expressed sequence tag (EST) GH06244 was tagged with HA at the C-terminus. Full length DMeltrin cDNA (CG31314, CG31385; GenBank accession number AY525769) was constructed from EST SD34743. The cDNA was HA-tagged at the C-terminus. Kuz dsRNA was generated by antisense 2626-3515, sense 2247-3515, DTACE and DMeltrin dsRNA constructs were generated from the pro-domain region in a similar way to ds-kul. All constructs were cloned into pUAST. UAS-Kuz plasmid (from D. Pan) was tagged at the C-terminus with HA.
Additional UAS lines used in cell culture assays were UAS-Myc-Delta (from K. Klueg), and UAS-Myc-Serrate (from R. J. Fleming).
Cell culture
Drosophila S2 cells were grown in Schneider's medium (Beit-Haemek)
with 10% heat-inactivated fetal calf serum. The calcium phosphate method was
used for transfection. To express the various constructs, UAS vectors were
co-transfected with the actin5c-GAL4 plasmid. Following transfection
the cells were grown in Schneider's medium without serum, and medium was
collected after 2-3 days. Cells were lysed in RIPA buffer. Protein extraction
and Western-blot analysis was performed as in Tsruya et al.
(Tsruya et al., 2002). For
Delta detection, Laemmli sample buffer without ß-mercaptoethanol was
used. UAS-GFP plasmid was used to calibrate the number of transfected cells.
Western blotting was carried out with anti-HA (Babco), anti-GFP (Roche), and
additional antibodies described below.
Immunohistochemistry and in-situ hybridization
Immunohistochemistry of larval discs was performed according to Tomlinson
and Ready (Tomlinson and Ready,
1987). Pupal wing staining was performed as in Axelrod
(Axelrod, 2001
). The following
primary antibodies were used: monoclonal C594.9B anti-Delta (1:100) (from S.
Artavanis-Tsakonas), rat anti-Serrate (1:1000) (from K. Irvine), and rabbit
anti-ß-Gal (1; 2000) (Cappel). The monoclonal antibodies 4D4 anti-Wg,
(1:10) and 2B10 anti-Cut, (1:10) were obtained from Developmental Studies
Hybridoma Bank. Secondary antibodies were obtained from Jackson
ImmunoResearch. In-situ hybridization on embryos and wing discs was performed
according to
www-biology.ucsd.edu/~davek/.
To generate antibodies against Kul, a kul cDNA fragment encoding amino acids 1215-1529 from the cytoplasmic tail was cloned into pRSETA. The resulting protein was injected into rats.
Sequence analysis
Phylogenetic analysis was performed using the extracellular region of the
ADAM proteins in the MEGA packages using the neighbor-joining method.
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Results |
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Among the five Drosophila metalloproteases, a single homolog was
identified for TNF- converting enzyme (TACE), two homologs were found
for Meltrin-
, and two for ADAM10. Analysis of the ADAM family
phylogenetic tree identified gene duplication events that took place most
probably after the divergence of the ancestors of nematodes and insects
(Fig. 1A). While Kuz shows a
high degree of similarity to human ADAM10, another Drosophila
protein, which we term Kuzbanian-like (Kul), exhibits an even higher degree of
similarity to ADAM10, especially in the disintegrin domain (which facilitates
substrate recognition), as well as in the metalloprotease catalytic domain
(Fig. 1B).
|
Serrate (Ser) is a second ligand of Notch in Drosophila and is employed in more restricted biological settings. A similar profile of cleavage was also observed for Ser, which was cleaved by Kuz, Kul and DTACE, but only marginally by DMeltrin (Fig. 1C). Detection of efficient cleavage in S2 cells, which are grown in suspension, supports the notion of cell-autonomous cleavage. Thus, cleavage of Dl or Ser by ADAM proteins is likely to take place within the same cell, rather than between adjacent cells.
Which ADAM proteins affect Notch signaling in the wing?
To examine the biological roles of the ADAM proteins that can cleave Dl, it
was necessary to compromise their activity in flies. Mutations or P-element
insertions in DMeltrin, DTACE and kul are not currently
available. We therefore generated double-stranded RNA (dsRNA) `knock-down'
constructs for each of these genes, directed against a region of minimal
similarity with the other family members.
Activation of the Notch pathway is required many times during normal wing
development. Especially notable is the role of Notch activation in restricting
the width of the wing veins within a pro-vein territory
(de Celis, 2003;
Huppert et al., 1997
).
Utilizing the UAS-GAL4 system, dsRNA constructs of Drosophila ADAM
metalloproteases were expressed in the wing. Expression of
ds-DMeltrin or ds-DTACE did not lead to any detectable wing
phenotypes, even when expressed under the regulation of the potent wing driver
MS1096-GAL4 (not shown). By contrast, induction of
ds-kul expression by the same driver gave rise to distorted wings
(not shown), and loss of the wing margin following induction by
sd-GAL4 (Fig. 7J).
Expression of ds-kul by the weaker driver, sal-GAL4,
resulted in two distinct adult wing phenotypes in the
spalt-expression domain, which encompasses the region between veins
L2-L4; formation of multiple wing hairs (A.S. and B.-Z.S., unpublished), and
partial loss of veins (Fig. 2B,
arrow). The first phenotype is Notch-independent, and will not be further
addressed in this work.
|
|
Characterization of Kul
In light of the high sequence similarity between Kul and Kuz, the
specificity and activity of ds-kul was verified in cultured cells.
When Kul was expressed in S2 cells, both a high molecular weight precursor
form, and a cleaved, mature form were detected. Expression of ds-kul
eliminated both forms of Kul, but did not affect Kuz protein levels,
demonstrating the specificity of ds-kul
(Fig. 2I).
The activity of ds-kul was also examined in vivo. A broad distribution of kul transcripts was detected during all embryonic stages (not shown) and in the wing imaginal discs (Fig. 2D). ds-kul expression in the wing reduced endogenous kul mRNA levels, illustrating the potency of ds-kul in vivo (Fig. 2E).
We further characterized the structural requirements for Kul function, by
examining in S2 cells protein maturation and the role of the different domains
in promoting Dl cleavage (Fig.
3). Basal cleavage of Dl was enhanced by co-expression of
full-length Kul. By contrast, a Kul variant bearing an E-to-A substitution
within the metalloprotease catalytic domain (E-A Kul), which abolishes
catalytic activity, failed to cleave Dl. This demonstrates the need for an
active protease domain in Kul. A similar mutation abolished the catalytic
activity of Kuz (Pan and Rubin,
1997).
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Kul regulates Notch signaling in the pupal wing veins
In the pupal wing, activation of the Notch pathway by Dl contributes to
refinement and restriction of the veins. Dl, expressed by the central pro-vein
cells, activates Notch in the lateral pro-vein domain, forcing these cells to
adopt an inter-vein fate, while the Dl-expressing cells themselves
differentiate as veins (de Celis,
2003) (Fig. 4E).
Reduction of Notch signaling causes lateral pro-vein cells to adopt a vein
cell fate, leading to vein thickening in the adult wing. By contrast, ectopic
expression of Dl by the inter-vein cells results in Notch activation in the
central pro-vein cells, leading to vein loss
(Huppert et al., 1997
).
|
Expression of ds-kul by sal-GAL4 had a marked effect on the expression of Dl, as well as on the Notch reporter (Fig. 4B,D). Irregular expansion of Dl into the lateral pro-vein territory was observed, while in other parts of the wing Dl expression disappeared from the veins. Notch-reporter expression expanded into the vein cells. Elevation in Dl levels in the lateral pro-vein cells endowed them with the capacity to activate Notch signaling within the vein, demonstrating a role for Kul in maintaining unidirectional signaling by the Notch pathway. These patterns account for the loss of veins seen in the adult wing following expression of ds-kul (Fig. 2B). The irregular patterns observed in the pupal wing may represent snapshots of a dynamic sequence, which is initiated by expansion of Dl to the lateral pro-vein cells, followed by expansion of Notch activation and loss of Dl expression in the vein cells.
Kul cleaves Delta in vivo in a cell-autonomous manner
The expansion of Dl-protein distribution in the pupal wing following
ds-kul expression implied that Kul normally contributes to the
restricted distribution of Dl in this tissue. To examine in more detail the
capacity of Kul to cleave Dl in vivo, we monitored the larval wing imaginal
disc. Dl protein, detected by an antibody recognizing the extracellular
domain, is normally observed as a membrane-associated protein that is elevated
in the vein and juxta-margin cells and excluded from the wing margin
(Fig. 5A)
(Kooh et al., 1993). We
monitored changes in Dl distribution in discs where Kul was overexpressed by
the MS1096 driver. Dl membrane-associated staining in the wing pouch
was diminished, and a residual punctate staining appeared, possibly reflecting
endocytosis of secreted Dl (Fig.
5B). Normal Delta distribution was retained in the notum, where
Kul was not overexpressed. Similarly, expression of Kul by sal-GAL4
eliminated Dl in the sal domain
(Fig. 5C,D). To verify that Kul
directly affects the cleavage of Dl, rather than Dl expression, we generated
clones overexpressing Dl under the regulation of actin-GAL4, in the
absence or presence of ectopic Kul. Indeed, the prominent appearance of Dl was
completely abolished when Kul was co-expressed in the same clone
(Fig. 5E-J).
|
|
In the wing disc, Notch signaling plays a key role in defining and
maintaining the margin, in two distinct signaling phases. First, the asymmetry
between the dorsal and ventral compartments defines the margin and induces the
expression of Wg by the future margin cells. The process is dictated by
expression of Fringe only in the dorsal compartment, facilitating Notch
signaling in the two cell rows comprising the border between the two
compartments (Fleming et al.,
1997; Neumann and Cohen,
1996
). The wing margin fate is subsequently maintained by
complementary unidirectional signals between the margin and juxta-margin
cells. In the margin, Notch signaling leads to the expression of Wg and Cut
(Neumann and Cohen, 1996
), the
latter operating as a transcriptional repressor of Dl. In parallel,
Wg activates expression of Dl and Serrate in the juxta-margin cells. High
levels of Dl and Serrate prevent Notch activation in these cells
(de Celis and Bray, 1997
;
Micchelli et al., 1997
). Thus
a stable loop of two reinforcing signals is generated
(Fig. 7K).
It was interesting to examine the biological consequences of the response
to Kul overexpression. Genetic removal of Dl or Ser alone was not sufficient
to alleviate the dominant-negative effect of the remaining ligand on Notch
signaling in the juxta-margin cells (de
Celis and Bray, 1997;
Micchelli et al., 1997
).
However, in the case of Kul overexpression, both ligands were affected, i.e.
Dl was efficiently cleaved and Ser was predominantly sequestered within the
cells. We observed an expansion in the expression of the Notch-target genes
wg and cut. In addition, the expression of Ser was broader
(Fig. 6C-G). We assume that
effective removal of Dl in conjunction with sequestration of Ser, gave rise to
alleviation of their dominant-negative effect on Notch signaling in the
juxta-margin cells expressing these ligands. Thus, a residual level of Dl or
Ser on the cell surface could trigger Notch signaling within these cells.
Activation of Notch subsequently leads to ectopic production of Wg, which in
turn spreads to neighboring cells to trigger ectopic expression of Ser.
Kul regulates Notch signaling in the wing margin
Does endogenous Kul in the wing disc have a role in maintaining the
asymmetric distribution of Dl? Expression of ds-kul by the potent
sd-GAL4 driver gave rise to loss of Cut and Wg expression in the
margin, and a reduction in the size of the wing pouch
(Fig. 7B,D). The adult wings
that developed were significantly reduced in size and showed no indication for
veins, and only rudimentary margin bristles in very restricted domains
(Fig. 7J). These results
demonstrate that Kul is essential for maintaining the spatial balance of Notch
signaling in the wing margin.
Induction of ds-kul by the sal-GAL4 driver did not affect the adult wing margin (Fig. 2B) and resulted only in a reduction in Wg levels in the wing margin (Fig. 7E,F), without a pronounced effect on Cut levels (not shown). In view of the role Kul plays in Dl cleavage, we wanted to test whether the changes in Notch target-gene expression in the wing margin resulted from elevation in Dl levels within the margin cells. Indeed, higher levels of Dl could be detected in the margin within the sal domain where ds-kul was expressed (Fig. 7I). By contrast, no effects of ds-kul on the distribution of Ser were observed (see Fig. S1 in the supplementary material).
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Discussion |
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It is interesting to note that, with respect to Dl cleavage, there is no
redundancy between the ADAM proteins that are able to cleave Dl in cell
culture. Removal of kul RNA alone was sufficient to give rise to
dramatic phenotypes. This feature is especially noteworthy with respect to Kul
and Kuz, which are both capable of cleaving Dl upon overexpression in the wing
disc. The requirement for Kul may reflect a quantitative aspect, i.e. the
activity of Kuz may not suffice to remove excess Dl. Alternatively, there may
be specific qualitative features to the removal of Dl by Kul. ADAM10 was also
shown to cleave Dl in cell culture (Six et
al., 2003). It is not clear what is the molecular basis for the
Notch-mutant phenotype ADAM10 knockout mice display
(Hartmann et al., 2002
),
because ADAM10 does not carry out the S2 cleavage of Notch
(Mumm et al., 2000
).
While naturally secreted versions of Notch ligands were recently identified
in C. elegans (Chen and Greenwald,
2004), it seems that in Drosophila cleavage of Dl does
not generate a biologically active form. Overexpression of cleaved Dl had no
detectable phenotypic consequences when expressed either in the eye or the
wing, two tissues in which Dl was shown to play a crucial role during many
phases (Mishra-Gorur et al.,
2002
).
Kul maintains unidirectional Notch signaling
We have demonstrated that the biological activity of Kul in removing Dl is
essential and non-redundant with other ADAM metalloproteases. In the absence
of this activity, the level of Dl in cells receiving the Notch signal is
elevated. As a result, the unidirectional signaling of Notch is skewed,
because the cells that normally receive the signal are converted to
signal-generating cells, and fail to respond to the normal cues presented by
Dl-expressing cells.
How does Kul activity impinge on the distribution of Dl? Overexpression of Kul in the wing disc resulted in a dramatic diminution of the levels of Dl. This effect is cell autonomous, i.e. Kul can only eliminate Dl within the cells in which it is expressed. It is not known if cleavage takes place once both proteins are localized to the cell surface, or if removal of Dl occurs during trafficking to the cell surface. Since no accumulation of Dl was observed within the cells following Kul overexpression, we favor the first possibility. Kul activity appears to be constitutive (see below), implying that there is no preferential cleavage of Dl by Kul in the receiving cells. Rather, the final outcome is likely to result from the activity of Kul in both cell types. In the receiving cells, where the levels of Dl are low, the proteolytic activity of Kul effectively eliminates the Dl protein. By contrast, in the sending cells expressing high levels of Dl, while Kul may cleave some of the ligand, sufficient levels of Dl remain to allow efficient signaling.
Disruption of Notch unidirectional signaling following removal of Kul
highlights the necessity of continuously removing the Dl protein, in order to
generate a setting in which it would be hard for the Dl protein to accumulate.
Transcriptional repression of Dl expression is not sufficient. For
example, in the wing margin, activation of the Notch pathway specifically
leads to the induction of E(spl) and Cut, which are transcriptional repressors
of Dl expression (de Celis and
Bray, 1997; Micchelli et al.,
1997
). Yet, in the absence of Kul, some Dl protein is produced by
the margin cells (Fig. 7I).
Similarly, in the pupal wing, activation of Notch in the lateral pro-vein
cells induces E(spl) expression (de Celis
et al., 1997
). Nevertheless, Dl is produced by these cells when
Kul is eliminated (Fig. 4D).
These observations underscore an inherent difficulty in shutting down
Dl transcription efficiently. They also imply that even residual
levels of Dl have detrimental biological consequences. The constitutive
cleavage of Dl by Kul is therefore a crucial safeguard, continuously removing
low levels of Dl that have escaped transcriptional repression.
The biological role of Kul was demonstrated in this work in two stages in
which Notch signaling refines a pre-existing asymmetry between adjacent cells:
the wing margin and the wing veins. In other instances, Notch signaling
actually generates the asymmetry between cells. Notch defines the correct
number and spacing of differentiated cells within a field of equipotent cells,
e.g. in the embryonic neuroectoderm or among pupal sensory organs. In these
cases, it is thought that stochastic fluctuations in the levels of Dl, coupled
to mechanisms that amplify these changes, lead to differentiation of some
cells and concomitant repression of differentiation in the neighboring cells
(Heitzler et al., 1996). Kul
does not seem to impinge on these process. No effects on the number and
organization of neuroblasts were observed following induction of
ds-kul by broad maternal and early zygotic drivers (not shown).
Another avenue of Notch signaling is triggered by asymmetric cell divisions in
the sensory neuron precursors
(Schweisguth, 2004
). Again,
induction of ds-kul by neu-GAL4 did not give rise to any
Notch-related phenotypes in the sensory bristles (not shown). We therefore
conclude that the activity of Kul appears to be essential for Notch signaling
specifically in cases where a pre-existing spatial asymmetry is used to guide
the directionality of Notch signaling.
Is Kul activity regulated?
In view of the central role of Kul in Notch signaling, it was important to
examine the different junctions in which Kul activity may be regulated. At the
transcriptional level, Kul appeared to be broadly expressed, in embryos and in
imaginal discs. This broad expression is also reflected in the
Notch-independent multiple-wing hair phenotype that was observed in all wing
cells where ds-kul was induced. It is still possible that the basal
level of Kul expression may be elevated in cells where Notch signaling takes
place, to reduce the levels of Dl in these cells more efficiently.
At the post-transcriptional level, however, there are several steps in the generation of an active Kul protein, which could be regulated. The protein must be correctly targeted to the plasma membrane, a process that may rely on the cytoplasmic domain of Kul and its interaction with the intracellular trafficking machinery. The precursor form of Kul undergoes processing by Furins, to remove the pro-domain. In the absence of this processing, Kul cannot cleave Dl. Finally, association of Kul with its substrates is mediated by the disintegrin domain, and possibly also by additional proteins that could bias this interaction.
In spite of the sequential processes necessary for the formation of a mature, active Kul protein, there is no evidence that any of these steps is regulated in time or space. The data so far support the notion of a constitutive maturation and processing of Kul. In every cell where Kul was misexpressed, an outcome was observed, as monitored by removal of Dl. In the wing, removal of Kul activity also gave rise to additional phenotypes that are not related to Notch, e.g. the appearance of multiple wing hairs (not shown). This phenotype was observed in all cells where ds-kul was expressed, again supporting the notion that Kul is normally expressed and activated uniformly.
In conclusion, while Kul may be broadly active, it enhances and maintains the asymmetrical activation of Notch, by relying on the initial differences in the levels of Dl. Kul effectively removes the ligand from the cells expressing Dl at low levels, while retaining sufficient levels of Dl in the cells that will activate Notch. Thus, a uniform activity of Kul can amplify a bias in the levels of Dl expression, and lead to a strict unidirectional activation of Notch, that is central to patterning the organism at multiple stages of development.
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Supplementary material |
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ACKNOWLEDGMENTS |
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