Delta
The Notch protein has 36 EGF-like repeats: two of them (numbers 11 and 12) are required for the interaction with the Delta and Serrate ligands. A Notch mutation has been isolated in its Delta- and Serrate-binding domain. It behaves genetically as both a Notch antimorphic and as a loss-of-function mutation. This mutation, NM1, carries a Glu to Val substitution in the Notch EGF repeat 12. The NM1 allele interacts with other Notch alleles such as Abruptex and split and with mutations in the Notch-ligand genes Delta and Serrate (deCelis, 1993).
During wing development in Drosophila, the Notch receptor is activated along the border between dorsal and ventral cells, leading to the specification of specialized cells that express Wingless (Wg) and organize wing growth and patterning. Three genes, fringe(fng), Serrate(Ser) and Delta (Dl), are involved in the cellular interactions leading to Notch activation. The relationship between these genes has been investigated by a combination of expression and coexpression studies in the Drosophila wing. Ser is normally expressed in dorsal cells while Dl is initially expressed by all wing cells. However, their expression soon becomes restricted to the dorso-ventral boundary. In order to study Ser and Dl signaling between dorsal and ventral cells, Dl and Ser were expressed ectopically along the anterior side of the anterior-posterior compartment boundary. These experiments confirm that Ser and Dl induce and maintain each other's expression by a positive feedback loop. Importantly, their ability to induce each others expression is dorsal-ventrally asymmetric, because Ser induces Dl strongly in ventral cells, but only very weakly in dorsal cells, whereas Dl induces Ser expression in dorsal cells, but not in ventral cells. fng is expressed specifically by dorsal cells and functions to position and restrict this feedback loop to the developing dorsal-ventral boundary. This is achieved by fng through a cell-autonomous mechanism that inhibits a cell's ability to respond to Serrate protein and potentiates its ability to respond to Delta protein (Panin, 1997).
To determine the effects of Fringe protein on Ser and Dl activity, margin gene expression and margin bristle formation were asayed while coexpressing these proteins in ventral cells. Ectopic expression of Ser leads to ectopic wing-margin gene expression and adult wing-margin formation in ventral cells along the edges of the ectopic Ser stripe. Misexpression of Fng in ventral cells inhibits these effects of Ser activity, demonstrating that Fng can inhibit Ser signaling. Misexpression of Dl induces ectopic wing-margin formation and Ser expression in dorsal cells but not in ventral cells. However, when Fng is misexpressed in ventral cells, Dl induces Ser expression and margin formation in both dorsal and ventral cells. Thus Fng potentiates Dl signaling, allowing ventral cells to respond to Dl just as dorsal cells normally do. Experiments show that Fng inhibits Ser activity only when it is expressd in receiving cells, and not when expression is restricted to Ser-signaling cells. Activated Notch can induce both Ser and Dl, and activated Notch has similar effects on dorsal and ventral cells, implying that Fng exerts its effects upstream of Notch activation. Because Fng is extracellular, this implies that activity of cell-associated Fng protein differentially modulates the binding and/or activation of Notch by its two ligands (Panin, 1997).
During investigations to further understand the biochemical mechanism by which Delta signaling is regulated, four Delta isoforms have been identified in Drosophila embryonic and larval extracts.
The proportions of these four isoforms vary during
embryonic development. The L isoform predominates in embryonic extracts during the first half of
embryogenesis, whereas three of the four isoforms are present in approximately equal amounts in
extracts prepared from animals completing embryogenesis. Immunoprecipitation of Delta from larval
extracts reveals that these four isoforms are present in relatively equal amounts.
In Drosophila cultured cells programmed to express full-length Delta protein, the Delta L I1, I2, and S
isoforms are found in cell extracts, and Delta S is also found in the surrounding medium. The apparent molecular weights of the embryonic and larval Delta S isoforms
are substantially less than that of the full-length L isoform; yet these Delta S isoforms react with
Delta-specific mAbs 9B and 8A, which are specific for the Delta extracellular domain.
Embryonic Delta S has an apparent molecular weight very similar to that of
DeltaSEC1, a secreted form of the Delta extracellular domain, truncated at amino acid 573, which has been expressed in Drosophila cultured cells. These findings suggest that Delta S could be
a proteolytically processed derivative of Delta L, cleaved within the Delta extracellular domain.
One or more proteolytic activities, present during later stages of embryogenesis and in young larvae, is shown to be capable of processing full-length Delta (Klueg, 1998).
Colocalization studies in cellular blastoderm embryos reveal that antibodies to the Delta intracellular domain localize to plasma membranes, as do antibodies to the Delta extracellular domain. However, some subtle differences at this stage in the distributions of Delta extracellular and intracellular
domain epitopes are observed. At high magnification, occasional foci of intense staining are observed at or near the plasma membrane with antibodies to the Delta extracellular domain. These foci are not detected with antibodies to the intracellular domain. Before
gastrulation, within the mesodermal anlage, antibodies to the intracellular domain localize to vesicular
structures as do antibodies to the extracellular domain. Merging these images
reveals that these two classes of Delta antibodies colocalize within a majority of these vesicular
structures. However, a few vesicles appear to react with only antibodies specific for
the extracellular or intracellular domain. Collectively, these localization studies in embryos and cultured
cells provide evidence for the existence and distinguishable localization of at least three Delta isoforms
in vivo: Delta L, Delta S, and Delta IC (Klueg, 1998).
Delta is demonstrated to be a transmembrane ligand that can be
taken up by Notch-expressing Drosophila cultured cells. Cell culture experiments imply that full-length Delta is taken up by
Notch-expressing cells. Evidence is presented that suggests this uptake occurs by a nonphagocytic mechanism. If phagocytosis or a similar mechanism mediates clearance of Delta-Notch complexes from the
surfaces of Notch+ cells during Delta-Notch interactions in S2 cells, one would predict that large
amounts of membrane from Delta+ cells would be taken up by Notch+ cells during this process. Vectors were used supporting expression of either of two full-length Drosophila transmembrane proteins,
Boss or Neuroglian, in cotransfections with an
expression vector encoding full-length Delta to mark Delta+ cell membranes. In
experiments in which Drosophila S2 cells are programmed to express full-length Delta and Boss, and
then aggregated with Notch+ cells, Boss is not detectable in Delta+ vesicles within Notch+ cells. However, on occasion, Boss colocalizes with Delta in Delta+ vesicles within
Delta+ cells. In similar experiments, using Neuroglian instead of Boss to label plasma
membranes, no colocalization of Neuroglian in Delta+ vesicles in Notch+ cells is observed (Klueg, 1998).
The finding of cross talk between two pathways is important news in developmental biology. Dishevelled is a key element in the wingless pathway, and also appears to physically interact with the intracellular domain of Notch. The
dishevelled gene interacts antagonistically with Notch and its ligand Delta. A direct physical interaction between Dishevelled and the Notch carboxyl
terminus, distal to the cdc10/ankyrin repeats, suggests a mechanism for this interaction.
It is proposed that Dishevelled, in addition to transducing the Wingless signal, blocks
Notch signaling directly, thus providing a molecular mechanism for the inhibitory cross
talk observed between these pathways (Axelrod, 1996).
The Notch signaling pathway plays an important role during the development of the wing primordium, especially of the wing blade and margin. In these processes, the activity of Notch is controlled by the activity of the dorsal specific nuclear protein Apterous, which
regulates the expression of the Notch ligand, Serrate, and the Fringe signaling molecule. The other Notch ligand, Delta, also plays a
role in the development and patterning of the wing. It has been proposed that Fringe modulates the ability of Serrate and Delta to signal through Notch and thereby restricts Notch signaling to the dorsoventral boundary of the developing wing blade. The results are reported of experiments aimed at establishing the relationships between Fringe, Serrate and Delta during wing development (Klein, 1998).
The initial stages of the development of the wing blade require Notch signaling and lead to the activation of the vestigial boundary enhancer (vgBE) at the
interface between dorsal and ventral cells. Ser is not involved in this event and therefore there ought to be another Notch ligand, under the control of ap, that
is responsible for the activation of the vgBE. The product of
the Delta gene is a good candidate for this function. During the second instar, Dl
is expressed throughout the wing disc, but it is slightly
upregulated over the ventral region and shortly afterwards, its
pattern of expression is identical to that of vgBE, i.e. a 2- to 3-
cell-wide stripe that straddles the DV interface. Furthermore,
the expression of Delta is similar to that of the vgBE in Ser and
ap mutant discs: in ap mutant wing discs, expression of Dl is
lost at the time when the wing primordium is induced, whereas in Ser mutants expression is detected until early third instar. This suggests that Delta might be
the activating ligand for the Notch-dependent expression of the
vgBE, which operates in the absence of Ser. Consistent with
this possibility, ectopic expression of Dl can
rescue the loss of wing blade tissue and of wing margin
characteristic of ap and Ser mutants (Klein, 1998).
Serrate, in turn, along with Delta, refines Notch function at the DV interface.
After the establishment and expansion of the wing primordium,
there is a new requirement for Notch signalling in the growth
and patterning of the wing blade. In this process, both Serrate
and Delta act as ligands for Notch and, as in earlier stages, have
different patterns of gene expression, which suggests that they might have
different functions. However, ectopic expression
of either Dl or Ser will rescue the loss of wing tissue and of
wing margin characteristic of ap mutants, and this raises the
question of why are there two different ligands to achieve the
activation of Notch in these early stages of wing development.
It is believed that coexpression of both ligands might result in a different degree of Notch activation than
would be achieved individually -- this might allow for a finer degree of regulation for Notch activity. In the
presence of Serrate, Delta is able to signal although with a reduced activity level, which perhaps reflects a competition between Serrate and Delta for Notch (Klein, 1998).
Cell-cell signaling mediated by the receptor Notch regulates the differentiation of a wide variety of cell
types in invertebrate and vertebrate species, but the mechanism for signal transduction following
receptor activation is unknown. A recent model proposes that ligand binding induces intracellular
processing of Notch; the processed intracellular form of Notch then translocates to the nucleus and
interacts with DNA-bound Suppressor of Hairless [Su(H)], a transcription factor required for target
gene expression. Intracellular cleaveage has been suggested to occur within either the transmembrane domin or the first 10 amino acids of the cytoplasmic domain. As intracellular processing of endogenous Notch has so far escaped
immunodetection, a sensitive nuclear-activity assay was devised to monitor indirectly the processing of
an engineered Notch in vivo. First, the non-membrane-tethered intracellular domain of Notch, fused to the
DNA-binding domain of Gal4, regulates transcription in a Delta-independent manner. This transcriptional regulation requires Su(H) activity, suggesting that Su(H) may not only target the Notch intracellular domain to the DNA but may also have an additional function. For instance, Su(H) may be required to protect processed Notch from degradation, or participate in transcriptional activation together with processed Notch. Subsequently, full-length Notch, containing the Gal4 DNA-binding domain inserted 27 amino acids
carboxy-terminal to the transmembrane domain, activates transcription in a Delta-dependent manner.
These results provide indirect evidence for a ligand-dependent intracellular processing event in vivo,
supporting the view that Su(H)-dependent Notch signaling involves intracellular cleavage, and
transcriptional regulation by processed Notch (Lecourtois, 1998).
Signaling by the Notch surface receptor controls cell fate determination in a broad spectrum of tissues.
This signaling is triggered by the interaction of the Notch protein with what, so far, have been thought to
be transmembrane ligands expressed on adjacent cells. Here biochemical and genetic analyses show that
the ligand Delta is cleaved on the surface, releasing an extracellular fragment capable of binding to Notch
and acting as an agonist of Notch activity. The ADAM disintegrin metalloprotease Kuzbanian is required
for this processing event. Given the similar phenotypes produced by loss of Notch signaling and loss-of-function mutations for kuz, it has been suggested that Kuz may be involved in the cleavage of N (Pan, 1996). This hypothesis is not corroborated by recent biochemical studies, indicating that the functionally crucial cleavage of N in the trans-Golgi network is catalyzed by a furinlike convertase (Logeat, 1997). These observations raise the possibility that Notch signaling in vivo is modulated
by soluble forms of the Notch ligands (Qi, 1999).
A genetic screen to identify modifiers of the phenotpes associated with the constitutive expression of a dominant negative transgene of kuz (kuzDN) in developing imaginal discs has identified Delta as an interacting gene (X. Wu, W. Wang, and T. Xu, unpublished observation reported by Qi, 1999). Flies expressing this dominant negative kuz construct, despite carrying a wild-type complement of kuz, become semi-lethal when heterozygous for a loss-of-function Delta mutation (X. Wu, W. Wang, and T, Xu, unpublished observation reported by Qi, 1999). In contrast, Delta duplications rescue the phenotypes associated with kuzDN. The kuzDN flies display extra vein material (especially deltas at the ends of the longitudinal veins); wing notching (observed with a low penetrance); extra bristles on the notum, and they also have small rough eyes. When kuzDN flies carry three, as opposed to the normal two, copies of wild-type Notch the bristle and eye phenotypes are not affected, nor are the vein deltas altered. However, the kuzDN phenotypes are effectively suppressed by Delta duplications, indicating that a higher copy number of Dl molecules is capable of overriding the effects of the kuzDN construct (Qi, 1999).
Delta has been shown to be cleaved by Kuz in transfected cultured cells, with the release of the extracellular domain of Delta. Sequencing reveals a putative propeptide processing site that is conserved in all Delta homologs. There is a distinct absence of cleaved Delta in kuz minus embryos but no difference in the processing of Notch. The biological activity of Delta extracellular domain can be detected in culture. Ligand-dependent Notch activation has been demonstrated in cortical neurons, which express endogenous Notch receptors, causing morphological changes as well as retractions of neurites. The same effects are observed when neurons are cultured in the presence of the extracellular domain of Delta. The importance of additional cleavages in Dl, the mode of activity of full-length Dl, and whether the second ligand Serate is also processed are critical questions to resolve. It is now apparent that future analysis of Delta in Notch signaling events must consider its potential as a diffusable ligand (Qi, 1999).
Molecular evidence has established that direct heterotypic
interactions occur between the Drosophila receptor Notch
and the ligands Delta and Serrate, and that homotypic
interactions occur between Delta molecules on opposing
cell surfaces. Using an aggregation assay developed for
Drosophila cultured cells, the affinities
of these interactions have been compared. The heterotypic
interactions between Notch and the ligands Delta and
Serrate have higher affinities than homotypic interactions
between Delta molecules. Contrary to previous suggestions,
the evidence implies that the interactions between Serrate
and Notch are similar in affinity to those between Delta and
Notch. Fringe does not detectably affect the
ligand-receptor interactions of the Notch pathway in
cultured cells. Furthermore, Serrate, like
Delta, is a transmembrane ligand that can participate in
reciprocal trans-endocytosis of ligand and receptor
between expressing cells. These findings imply that
qualitative differences between Delta- and Serrate-mediated
Notch signaling depend on characteristics other
than intrinsic ligand-receptor affinities or the ability to
participate in reciprocal ligand and receptor trans-endocytosis (Klueg, 1999).
The Delta extracellular domain and intracellular domain are taken up by adjacent Notch+ Drosophila cultured
cells. Using antibodies against the Serrate extracellular domain
or a MYC epitope tag in the intracellular domain, it was asked
whether the entire Serrate molecule is transferred into
neighboring Notch+ cells. In Drosophila Serrate-Notch cell
aggregates, the Serrate extracellular domain and
intracellular domain are found to be taken up by neighboring Notch+ cells. The fraction of Notch+ cells
adjacent to one or more SerrateWTMYC+ cells that contain
vesicles positive for the Serrate extracellular domain is 17%. This is similar to the fraction of Notch+ cells that
contain vesicles positive for the Serrate intracellular domain
(15%). The fraction of Notch+ cells that contain
vesicles positive for the Delta extracellular domain (27%) was
recorded in parallel as a control (Klueg, 1999).
Delta+ cells adjacent
to Notch+ cells contain vesicles positive for the Notch
intracellular domain, demonstrating that the entire Notch
molecule can be taken up by adjacent Delta+ cells. To determine whether similar trans-endocytosis
occurs during Serrate-Notch interactions, trans-endocytosis
of Notch into SerrateWTMYC+ cultured cells was examined. Notch is found to be trans-endocytosed, and the
percentage of SerrateWTMYC+ cells that contain vesicles
positive for the Notch intracellular domain is 38%, similar to
the percentage of Delta+ cells that contain vesicles positive for
the Notch intracellular domain. Heterotypic aggregates formed after 15 minutes of aggregation
were examined, and it was found that trans-endocytosis of Delta and Notch occurs
shortly after their interaction is initiated. Similar observations have been repeated for Serrate+-Notch+ heterotypic cell aggregates. Both Serrate and Notch are
trans-endocytosed shortly after interaction is initiated. Delta, however, is not taken up by adjacent Delta+
cells in homotypic aggregates (Klueg, 1999).
Receptor and ligand processing have recently come under
scrutiny as critical elements in the regulation of Notch
signaling. Delta is proteolytically processed to yield at least
four isoforms; however, the functional
significance of this processing is currently unclear. Processing
of Delta may be necessary for signal activation
or for downregulation, or may result from
protein degradation following clearance of ligand from the cell
surface. Notch is processed in a complex manner that is
thought to be required for genesis and activation of the receptor. First, Notch is cleaved during transport
through the Golgi at a site amino-proximal to the
transmembrane domain ('site 1' or 'S1') by a furin-like
convertase. Following this cleavage,
Notch is 'reassembled' and transported to the cell surface as a
heterodimeric receptor. Another cleavage event (termed 'S3') within the
intracellular domain has also been shown to occur. The S3
cleavage of Notch is ligand-dependent and produces a Notch
intracellular domain fragment that may act, in conjunction with
Su(H), in the nucleus as the primary Notch signal transducer. The mechanism that triggers the intracellular domain
cleavage is unknown. However, the Notch/lin-12 repeats
(LNRs) within the receptor extracellular domain may
contribute to regulation of this cleavage. When the LNRs are removed, intracellular
domain cleavage occurs in a constitutive manner in the absence
of ligand. This has led to the hypothesis
that binding of Delta to Notch may result in a cleavage event
(termed 'S2') in the Notch extracellular domain. This cleavage
would uncouple the LNRs from the remainder of the receptor,
allowing the intracellular domain cleavage (S3) to occur
constitutively. Recently, Notch S2 cleavage has been
demonstrated in mammalian cells (Mumm, 2000 in press, cited in Parks, 2000). S2
cleavage in these cells occurs in response to ligand binding and
blocking S2 cleavage results in loss of S3 cleavage, consistent
with a proteolytic cascade model of Notch activation (Parks, 2000).
Endocytosis of the ligand Delta (by the signaling cell), possibly by inducing cleavage of the
receptor at the S2 site, is required for activation of
the receptor Notch during Drosophila development. The
Notch extracellular domain (NotchECD) dissociates
from the Notch intracellular domain (NotchICD) and is
trans-endocytosed into Delta-expressing cells in wild-type
imaginal discs. Reduction of dynamin-mediated
endocytosis in developing eye and wing imaginal discs
reduces Notch dissociation and Notch signaling.
Furthermore, dynamin-mediated Delta endocytosis is
required for Notch trans-endocytosis in Drosophila
cultured cell lines. Endocytosis-defective Delta proteins fail
to mediate trans-endocytosis of Notch in cultured cells, and
exhibit aberrant subcellular trafficking and reduced
signaling capacity in Drosophila. It is suggested that
endocytosis into Delta-expressing cells of NotchECD bound
to Delta plays a critical role during activation of the Notch
receptor and is required to achieve processing and
dissociation of the Notch protein (Parks, 2000).
The separation of NotchECD from
NotchICD would relieve LNR-mediated repression of the S3
cleavage, which would then occur constitutively to release a
non-membrane bound, activated form of NotchICD. Several
predictions of this model are borne out. Dl alleles that encode
endocytosis-defective ligands are loss-of-function mutations,
and these defective ligands fail to support Notch trans-endocytosis
in cultured cells and Delta-mediated signaling in vivo. Dynamin function, which is necessary for Notch
signaling, is required for Delta endocytosis and for Notch
trans-endocytosis in cultured cells, and for dissociation of
NotchECD from NotchICD in developing imaginal tissues. In
addition, the third epidermal growth
factor-like repeat within the Delta extracellular domain is
required for Delta endocytosis and Notch trans-endocytosis,
and for Delta-dependent signaling during development (Parks, 2000).
Three alternative mechanisms are proposed by which
endocytosis may induce receptor activation. (1) After
binding of Delta to Notch, molecular strain imparted to Notch
by endocytosis in the signaling and receiving cells results in
a conformational change that permits access by processing
enzyme(s) to the S2 site. (2) The S2 site is masked by proteins
that interact with Notch to form a complex. Following Delta
binding, endocytosis of Delta and Notch alters intramolecular
interactions within the complex, unmasking the S2 site and
making it available for cleavage. (3) Endocytosis is not
required for Delta-induced S2 cleavage, but is instead required
to separate NotchECD and associated proteins from the
remainder of the Notch protein, thus relieving inhibition of S3
cleavage by NotchECD. The fact that Serrate-expressing
cultured Drosophila cells mediate Notch trans-endocytosis
at frequencies similar to those observed for Delta-expressing
cells suggests that
trans-endocytosis of NotchECD is one aspect of the Notch
activation mechanism that is common to Notch ligands (Parks, 2000).
The activation of Notch is regulated both by the temporal
and spatial distribution of the ligands and by the expression
of proteins such as Fringe (Fng) that are able to modulate
ligand-receptor interactions. This was
first evident in the developing wing, where Notch activity
results in the expression of genes such as wingless and
cut in a narrow 2- to 4-cell-wide domain at the
dorsoventral boundary. In this process, Fng
influences the effectiveness of the interactions between Notch
and its ligands by preventing Ser-mediated activation and
potentiating Notch activation by Dl. The
localized activation of Notch initially occurs because Apterous
promotes the expression of both Ser and Fng in dorsal cells,
while the inhibitory effect of Fng on Ser/Notch restricts Ser
signaling primarily to ventral cells. At the same time, the effect of Fng on
Dl has the consequence that ventral Dl-expressing cells signal
primarily to dorsal cells. A similar process occurs in the eye, where again the
compartment-specific expression of fng allows localized
activation of Notch at the eye dorsoventral boundary (de Celis, 2000 and references therein).
Conventionally Dl and Ser are considered activating ligands
of Notch and, in many instances, their elimination has non-autonomous
effects on development that are characteristic of a
membrane-associated ligand.
However, in the Drosophila wing and eye, both Notch ligands
have also been shown to have cell-autonomous inhibitory
effects on the activity of the receptor. Thus, the elimination of both ligands
in clones of cells in the wing can result in Notch activation
within the clone, detected as ectopic ct expression, indicating
that a normal function of Dl and Ser is to prevent Notch
activation within the cells in which they are expressed. In addition, ectopic expression of Dl
or Ser in groups of cells causes Notch activation only in the
adjacent cells. Consistent with the
suggestion that the inhibitory activity of the ligands relies on
interactions occurring between molecules within the same cell,
the negative effects of ectopically expressed Ser can be
alleviated by co-expression of full-length Notch. The negative effect of the ligands
could be instrumental in determining the polarity of Notch
signaling: cells expressing higher levels of ligand would have
reduced Notch responsiveness compared to adjacent cells with
lower ligand levels and hence Notch would be more readily
activated in the cells with relatively less ligand. The concept
that the relative levels of Notch and Dl are important for
signaling is also evident from the phenotypes caused by
varying the dosage of these genes. Finally, Dl
and Notch have been seen to co-localize on the surface of
cultured cells, suggesting that they could interact in the plasma
membrane. However, the antagonistic
interactions could be occurring anywhere within the cell and
the functional domain of Notch involved in this process has not
been characterised (de Celis, 2000 and references therein).
The extracellular domain of Notch contains an array of 36
EGF repeats, two of which, repeats 11 and 12, are necessary
for direct interactions between Notch with Delta and
Serrate. An investigation has been carried out of the function of a region of the
Notch extracellular domain where several missense
mutations, called Abruptex, are localized. These Notch
alleles are characterized by complex complementation
patterns and phenotypes that are the opposite of those observed with a loss
of Notch function. In Abruptex mutant wing discs, only the
negative effects of the ligands and Fringe are affected,
resulting in the failure to restrict the expression of cut and
wingless to the dorsoventral boundary. It is suggested that
Abruptex alleles identify a domain in the Notch protein that
mediates the interactions between Notch, its ligands and
Fringe that result in suppression of Notch activity (de Celis, 2000).
In wild-type discs, the response of
Notch to Dl and Ser is affected by the
presence of Fng, which is expressed in
dorsal cells. Since the domain of Fng
expression corresponds to the region
where Dl loses its capacity to
antagonize Notch in NAx mutants, an analysis was carried out to see
whether NAx mutations have
an altered sensitivity to Fng by
comparing the consequences of ectopic
fng expression in wild-type and NAx
discs. As with the ectopic ligand
expression, clones of cells expressing
fng that cross the dorsoventral
boundary inhibit expression of ct
except at the clone borders.
When the Fng-expressing clones lie in
the ventral compartment, ct is induced
in the cells at the boundary of the
clone, with the result that ct is detected
in neighboring fng+ and fng- cells. The ability of Fng to prevent ct
expression is reduced when Fng-expressing
clones are induced in NAx
mutant backgrounds. In a weak NAx allelic combination,
the expression of ct is still highest at clone boudaries, but
significant expression is detected within the clone. In the more severe mutants,
the Fng-expressing cells have little or no inhibitory effect on
ct, and there are high levels of Ct throughout the clone. Similar effects are seen when fng misexpression
is driven by Gal4-sal. Normally this causes an inhibition of ct
expression at the dorsoventral boundary; in NAx mutant discs,
however, Ct is detected throughout most of the domain of
ectopic Fng-expression.
If the NAx domain is significant in the interactions between
Notch and Fng, the NAx mutations should modify phenotypes
caused by alterations in fng expression. In the allele fngD4, fng is expressed throughout the wing pouch, causing severe scalloping of the wing margin. This correlates with the loss of ct and wg expression at the dorsoventral boundary and the expansion of vvl/drifter
expression. In NAx
heterozygous flies, the phenotype of fngD4 is reduced both at
the level of wing scalloping and the expression of dorsoventral
boundary markers. In hemizygous NAx males, both the expression of ct
and vvl and the adult phenotype are similar to the expression and phenotype typical of
NAx. Taken together, these results suggest that
NAx proteins are also deficient in some activity related to the
capability of Fng to restrict Notch activity (de Celis, 2000).
The amino-acid sequence of Fng indicates that it could be
a glycosyltransferase. Since NAx mutations
affect the extracellular domain of Notch,
the fact that the NAx alleles have altered behavior with respect
to Fng suggests that the mutated domain could be a target for
Fng-mediated glycosylation. If the NAx mutations perturb
glycosylation of Notch by Fng, this might explain why they
only affect the activity of Notch in the imaginal discs and not
in the early embryo, since fng is only required at later stages
of development. NAx alleles also affect several processes, such
as sensory organ development and vein cell differentiation, that
do not seem to require fng activity. This indicates that the NAx domain also affects
negative interactions between Notch with Dl and Ser
independent of fng function (de Celis, 2000).
The results shown here indicate that the NAx domain of Notch
is only necessary to mediate the functions of Fng and the ligands
that result in the suppression of Notch activity. A comparison
between the effects on Fng, Dl and Ser indicates that the
interactions between these molecules and Notch are affected to
different extents by NAx mutations. For example, although the
dominant negative effects of Dl and Fng are dramatically
reduced in NAx alleles, these mutations do not appear to
compromise the potentiating effect of Fng on Dl activation,
since there is still a strong bias towards Dl activity in the dorsal
domain where Fng is present. Similarly high levels of ectopic
Ser can efficiently suppress Notch activity in NAx backgrounds,
even though the phenotype of NAx mutant discs indicates that
NAx mutations perturb the dominant negative effects of Ser
when it is expressed at normal levels. Each NAx allele has a
characteristic strength that is reflected in its phenotype and
in the extent of ectopic ct activation. Furthermore, heteroallelic
combinations between NAx alleles often result in synergistic
phenotypes, a phenomenon called negative complementation. This suggests that the correct
conformation of the NAx domain in Notch multimers is critical
for the efficiency of the interactions between Notch, its ligands
and Fng that determine suppression of Notch activity (de Celis, 2000).
The Delta and Serrate proteins interact with the
extracellular domain of the Notch receptor and initiate
signaling through the receptor. The two ligands are very
similar in structure and have been shown to be
interchangeable experimentally; however, loss of function
analysis indicates that they have different functions during
development and analysis of their signaling during wing
development indicates that the Fringe protein can
discriminate between the two ligands. This raises the
possibility that the signaling of Delta and Serrate through
Notch requires different domains of the Notch protein.
This possibility has been tested by examining the ability
of Delta and Serrate to interact and signal with Notch
molecules in which different domains have been deleted.
This analysis has shown that EGF-like repeats 11 and 12,
the RAM-23 and cdc10/ankyrin repeats and the region C-terminal
to the cdc10/ankyrin repeats of Notch are
necessary for both Delta and Serrate to signal via Notch.
They also indicate, however, that Delta and Serrate utilize
EGF-like repeats 24-26 of Notch for signaling, but there
are significant differences in the way they utilize these
repeats (Lawrence, 2000).
Expression of Delta and Serrate with
molecules that lack EGF-like repeats 24-26 produce differing
results. Delta can signal through a Notch protein that lacks these
repeats, but Serrate cannot. This requirement for EGF-like
repeats 24-26 is surprising and indicates that the influence of
these repeats on the interactions between Notch and its ligands
in cell culture and in
vivo might reflect a requirement for these repeats
in signaling. Interestingly, there are differences in the way
Delta and Serrate require these repeats. In the case of Serrate,
the requirement for signaling is absolute, whereas in the case
of Delta it is conditional on the presence of EGF-like repeats
17-19, which have been shownto be structurally related to 24-26.
These differences are made more clear when comparing the
interactions of Delta and Serrate with the dominant negative extracellular Notch constructs (ECNs). Delta cannot
signal with full length Notch molecules that lack repeats 10-12 or 17-19
and 24-26, nor interact with ECN molecules that lack these
repeats. Although Serrate cannot signal with
FLN molecules lacking these repeats, it can, however, interact with the
corresponding ECN molecules. This suggests that whereas in
the case of Delta a direct interaction with Notch amounts to
signaling, this might not be sufficient for Serrate. In the case of Serrate, further modifications
of Notch or interactions with other proteins might be required
to trigger a signal (Lawrence, 2000).
Notch, a cell surface receptor, is required for the production of different types of cells during Drosophila development. Notch activates expression of one set of genes in response to ligand Delta and another set of genes in response to the
ligand Wingless. Just how Notch initiates these different intracellular activities has been the focus of this study. Cultured
cells expressing Notch were treated with Delta or Wingless, and the effect on Notch was examined by Western blotting.
Treatment of cells with Delta results in accumulation of ~120-kDa Notch intracellular domain molecules in the cytoplasmic
fraction. This form of Notch does not accumulate in cells treated with Wingless; rather, the ~350-kDa full-length Notch molecules accumulate. These results indicate that
N responds differently to binding by Delta and by Wingless, and suggest that although the Delta signal is transduced by the Notch intracellular domain released from the
plasma membrane, the Wingless signal is transduced by the Notch intracellular domain associated with the plasma membrane. It is proposed that the N receptor is a 'switch' for activation
of different signaling pathways during development. Dl binds the EGF-like repeats 11-12 region to shunt the N120-Su(H) complex into the nucleus for
turning on the expression of Dl-related genes. Wg binds the EGF-like repeats 19-36 region to send a transcriptional activator to the nucleus for turning on the
expression of Wg-related genes (Wesley, 2000).
Schneider cells expressing Notch (S2-N cells) treated with Dl for 1h accumulate ~120-kDa N molecules (N120). Dl binds N in the extracellular region, including EGF-like repeats 11 and 12). S2 cells expressing N molecules
lacking this region, NDeltaEGF1-18, do not accumulate N120 molecules in response to treatment with Dl. This indicates that N120 accumulates in response to Dl binding N. N120 is the complete intracellular domain and is
similar to the ~120-kDa N intracellular domain molecule shown to accumulate in vivo in response to D (Wesley, 2000).
N120 molecules do not accumulate in S2-N cells treated with Wg for 1, 2, or 5 hours. However, S2-N cells treated
with Wg for 5h accumulates ~350-kDa N molecules (N350) but not S2-N cells treated with Dl. N350 is the full-length co-linear N molecule
containing both the intracellular and extracellular domains. Wg binds N in the EGF-like repeats 19-36 region. S2 cells expressing N molecules lacking this
region, NDeltaEGF19-36, do not accumulate co-linear molecules when treated with Wg for 5h. In contrast, truncated,
co-linear NDeltaEGF19-36 molecules containing the Wg binding sites accumulate upon treatment with Wg. These results indicate
that accumulation of N350 in S2-N cells is in response to Wg binding N. Accumulation of N350 molecules is also discernible in cells treated with Wg for 2h
when the blots are exposed to film for shorter periods. In contrast to Wg-treated cells, Dl-treated cells in the same blots always have lower levels of N350 compared
with the levels in untreated cells (Wesley, 2000).
Accumulation of N350 molecules in Wg-treated cells is not due to activity of the endogenous Notch gene, which is rearranged in S2 cells. It is not due to a
general increase or stabilization of all proteins in the cells: all N molecules do not accumulate, and the total protein levels in the three lanes are comparable. It is also not due to a Wg effect that is unrelated to N binding but
retards N processing for cell surface presentation. Otherwise, co-linear NDeltaEGF19-36 would have also
accumulated, but it did not. Thus, whereas Dl binding full-length N results in accumulation of N120, Wg binding results in accumulation of the co-linear N350 (Wesley, 2000).
Treatment of S2-N cells with Dl or Wg for 2 h also results in accumulation of ~55-kDa N molecules (N55). N55 contains only the amino
terminus half of the intracellular domain, requires about 2 h to accumulate, and is variably recovered after about 3 h of treatment (Wesley, 2000).
To determine whether the responses observed in S2 cells are general N responses to treatments with Dl and Wg, the experiments were repeated with clone-8 cells
that express N endogenously. The results show that N in clone-8 cells responds similarly to N in S2 cells. Treatment with Dl results in accumulation of
N120 and not N350, whereas treatment with Wg results in accumulation of N350 and not N120; both Dl and Wg treatments result in accumulation of N55
molecules. The difference in levels of N350 between Dl-treated and Wg-treated cells is obvious after just 2 h of treatment. Clone-8 cells express a
higher level of N55 molecules in the absence of any treatment, presumably because they also express Dl endogenously (Wesley, 2000).
When Dl binds N in vivo, the ~120-kDa N intracellular domain is released into the cytoplasm. To determine whether the N120 in these in vitro
experiments with Dl also accumulates in the cytoplasm, S2-N cells were fractionated and analyzed following treatments with Dl and Wg. Following treatment with Dl,
N120 molecules accumulate in the cytoplasmic fraction. In contrast, N350 molecules accumulate in the membrane fraction following treatment with Wg. N55 molecules are not consistently detected in these experiments as they are very unstable in this fractionation and extraction procedure (Wesley, 2000).
It is not known whether the N120 molecules that accumulate in the cytoplasm in response to Dl are the same as those present in the membranes or
whether they are different molecules migrating in the same region of the gel. Membrane-tethered N intracellular domain (Nintra), untethered Nintra, and N120 migrate
alongside each other in these gels. N120 molecules associated with the membranes or with the cytoplasm are probably the membrane-tethered or released N
intracellular domain, respectively. Accumulation of N350 molecules in response to Wg is likely to be in the intracellular membranes associated with production of the
heterodimeric cell surface receptor. N55 is derived from N350 upon activation of Notch signaling by a ligand (Wesley, 2000).
Cellular signaling activities must be tightly regulated for proper cell fate control and tissue morphogenesis. The
Drosophila leucine-rich repeat transmembrane glycoprotein Gp150 is required for viability, fertility and development of the eye, wing and
sensory organs. Gp150 might function in subcellular vesicles to control appropriate intracellular levels of Dl
to modulate N signaling. In the eye, Gp150 plays a critical role in regulating early ommatidial formation. Gp150 is highly expressed in cells of the
morphogenetic furrow (MF) region, where it accumulates exclusively in intracellular vesicles in an endocytosis-independent manner. Loss
of gp150 function causes defects in the refinement of photoreceptor R8 cells and recruitment of other cells, which leads to the formation
of aberrant ommatidia. Genetic analyses suggest that Gp150 functions to modulate Notch signaling. Consistent with this notion, Gp150 is
co-localized with Delta in intracellular vesicles in cells within the MF region and loss of gp150 function causes accumulation of intracellular Delta protein. Therefore,
Gp150 might function in intracellular vesicles to modulate Delta- Notch signaling for cell fate control and tissue morphogenesis (Fetchko, 2002).
Sequence analysis indicates that the gp150 gene consists of six exons and five introns. The gp150 open reading frame (ORF) is restricted to the last four exons, which encode a polypeptide of 1051 amino acids. The extracellular domain of Gp150 contains 18 LRR motifs that might provide a scaffold for mediating protein-protein interactions. In
addition, it has several unique structural features that are conserved during evolution. These features include specific potential N-glycosylation
sites, cysteine motifs and acidic regions that flank the LRR region. Although the intracellular domain is short, it contains three conserved tyrosine phosphorylation
motifs that are potential targets of tyrosine kinases and receptor tyrosine phosphatases. The conserved tyrosine phosphorylation motifs might be used for interaction with SH2 domains of some docking proteins (Fetchko, 2002).
Phenotypic analysis has demonstrated that Gp150 plays a critical role during ommatidial development. In the absence of gp150 function, both the selection and patterning of the R8 cells become aberrant and a small number of ectopic R8 cells appear to be specified. The gp150 mutant eye phenotypes mimic what occurs in loss-of-function mutants of Dl and N, suggesting that Gp150 might facilitate Dl-N signaling. Supporting this hypothesis, the reduction of Dl or N function dominantly enhances the gp150 mutant eye phenotypes (Fetchko, 2002).
Clonal analysis was carried out to reveal in which cells the function of Gp150 is required. By examining phenotypically normal but genetically mosaic ommatidia, it was found that gp150 could be either wild type or mutant in all R cells. Therefore, the function of gp150 does not appear to be required in R cells for normal ommatidial development. Given that R cells are signal-sending cells, which prevent neural differentiation in neighboring precursor cells, this result is consistent with a view that gp150 might be required in cells that function as precursor cells to R cells (Fetchko, 2002).
How might Gp150 modulate Dl-N signaling? So far, Gp150 is the only protein known to co-localize with Dl in intracellular vesicles. In a simple model, Gp150 might facilitate Dl presentation at the cell surface. Gp150 might also promote fusion between Dl-positive MVBs and lysosomes so that the intracellular levels of Dl can be reduced. The latter mechanism might be used to reduce the autonomous inhibitory effect of Dl on N signaling in the signal-receiving cells. In the absence of gp150 function, the levels of intracellular Dl increase, which autonomously blocks the N pathway in N signal-receiving cells and possibly makes these cells more responsive to instructive signals for R cell specification and differentiation, thus resulting in ommatidia with too many R cells. In tissues such as the wing and sensory organs, Dl has been shown to autonomously inhibit N signaling in a signal-receiving cell. In some cases, increased intracellular levels of Dl might allow some Dl to be presented at the cell surface to initiate N signaling on adjacent cells. This would increase lateral inhibition and result in the formation of ommatidia with fewer R cells. The fewer R cell phenotype could also be due to an insufficient number of competent retinal precursor cells that are available for the competing ommatidial clusters during R cell recruitment in gp150 mutants. In any event, the possibility that Gp150 might use other mechanisms to modulate Dl-N signaling cannot be excluded. For instance, the up-regulation of Dl in gp150 mutant eye discs might be due to impaired N signaling, since N signaling can often cause down-regulation of Dl expression in signal-receiving cells. Further work is needed to distinguish among these possibilities (Fetchko, 2002).
N signaling is known to be regulated through mechanisms such as site-specific proteolysis and glycosylation. In addition, N is recognized and ubiquitylated by an E3 ubiquitin ligase Itch, suggesting that N stability could be regulated through the proteasome-mediated pathway. An Itch-related Drosophila protein, Suppressor of deltex, also negatively regulates N signaling. Another negative regulator of N, Numb, has recently been shown to be an endocytic protein, suggesting that Numb regulation of N could occur in the endocytic pathway. The regulation of Dl is also complex. For instance, Dl is proteolytically processed so its extracellular domain can be released. From this work, it is proposed that Gp150 might function in intracellular vesicles, which include endosomes, to adjust appropriate intracellular levels of the Dl protein for modulating N signaling (Fetchko, 2002).
Functional analysis of Gp150 indicates that both the extracellular and intracellular domains of Gp150 are essential. The extracellular domain of Gp150 contains 18 LRR motifs that might provide a scaffold for mediating protein-protein interactions. In addition, it has several unique structural features that are conserved during evolution. These features include specific potential N-glycosylation sites, cysteine motifs and acidic regions that flank the LRR region. Although the intracellular domain is short, it contains three conserved tyrosine phosphorylation motifs that are potential targets of tyrosine kinases and receptor tyrosine phosphatases. The conserved tyrosine phosphorylation motifs might be used for interaction with SH2 domains of some docking proteins. Further studies are required to reveal the functional significance of these conserved structural motifs, which should help reveal mechanisms of Gp150 action in cell fate control and tissue morphogenesis (Fetchko, 2002).
The receptor protein Notch is inactive in neural precursor cells despite neighboring cells expressing ligands. Specification of the R8 neural photoreceptor cells, which initiate differentiation of each Drosophila ommatidium, was investigated. The ligand Delta was required in R8 cells themselves, consistent with a lateral inhibitor function for Delta. By contrast, Delta expressed in cells adjacent to R8 could not activate Notch in R8 cells. The split mutation of Notch was found to activate signaling in R8 precursor cells, blocking differentiation and leading to altered development and neural cell death. split does not affect other, inductive functions of Notch. The Ile578-->Thr578 substitution responsible for the split mutation introduced a new site for O-fucosylation on EGF repeat 14 of the Notch extracellular domain. The O-fucose monosaccharide did not require extension by Fringe to confer the phenotype. These results suggest functional differences between Notch in neural and non-neural cells. R8 precursor cells are protected from lateral inhibition by Delta. The protection is affected by modifications of a particular EGF repeat in the Notch extracellular domain. These results suggest that the pattern of neurogenesis is determined by blocking Notch signaling, as well as by activating Notch signaling (Li, 2003).
N signaling in response to Dl is patterned in two
distinct ways. In some situations, typified by induction of the wing margin, the expression pattern of Dl contributes to where N will be activated. N remains inactive where Dl is not expressed. In other cases, typified by lateral specification of R8 precursor cells during eye development, N and Dl are expressed homogeneously, and the pattern of N signaling depends on differential activity of the N and Dl proteins. Even though Dl is expressed homogeneously, it is essential in the cells taking R8 precursor fate. The requirement for Dl in the R8 precursor cannot be substituted by Dl expression in the other cells, even though together they contact all of the cells that the R8 precursor contacts. This suggest that the interaction between Dl in cells selected for R8 precursor fate and N in other cells might be
qualitatively different from any interaction between Dl on non-R8 cells and N in R8 precursor cells (Li, 2003).
Inactivity of N in R8 precursor cells is not a passive event defined by absence of ligands, because even ubiquitous Dl overexpression fails to activate N in R8 precursor cells. By contrast, a recessive mutation, the split allele of N, now permits N to be activated by Dl in R8
precursor cells but has little or no effect on N signaling in many other contexts. The Dl protein in non-R8 cells is in an active form, because it can activate R8-cell N in the spl mutant (Li, 2003).
The spl mutant affects development of many retinal cell types. There is an R8 cell deficit, many other retinal cells are missing, cell death is elevated and additional cells may take R7 fate. The initiation and maintenance of atonal expression is deficient even before R8 specification begins. Mosaic analysis demonstrates that all these defects depend on the genotype of R8 cells only. Therefore N is activated in spl mutant R8 cells. Other cells must be affected indirectly as a consequence of the abnormal R8 cells. In confirmation of this, activation of the N signal
transduction pathway solely in R8 cells recapitulates the spl
phenotype, including the effects on other cell types (Li, 2003).
The notion that many cells might be affected indirectly in spl mutants is consistent with the role of R8 cells in founding each ommatidium. R8 cells initiate the cascade of EGF receptor-mediated inductions that recruit most of the retinal cell types, and are required for the survival of
unspecified cells. The effectiveness with which R8 cells carry out these roles depends on the level of atonal expression in the R8 precursors. Reduced Atonal expression in the ato2 mutant, which is defective in ato autoregulation, reduces recruitment of other cell fates because EGF receptor is activated in fewer surrounding cells. Elevating Atonal expression by targeted expression in R8 using the G109-68 driver leads to activation of EGF receptor in more cells than normal and recruitment of excess outer photoreceptor cells. Thus, losses of many other cells are an expected consequence of the reduced Atonal expression that occurs in spl mutant R8 cells (Li, 2003).
In addition to producing ligands for the EGF receptor, R8 and other
photoreceptor cells also secrete Hh, the primary signal moving the
morphogenetic furrow across the eye disc. Reducing
Atonal levels in R8 has further phenotypic effects through altered Hh
signaling. It is proposed that defective Hh signaling is the likely
explanation of non-autonomous effects of spl on the initiation of atonal expression in the morphogenetic furrow (Li, 2003).
The spl mutation also affects differentiation of sensory bristles
in the epidermis. As in R8 cells in the eye, sensory organ precursor cells
are specified by lateral inhibition but not inhibited by ectopic Dl expression. N signaling is important in
cell fate specification within the lineage of cells descended from sensory
organ precursors. It is plausible that aberrant N signaling might be
responsible for bristle defects in spl mutants, although this has not been examined directly (Li, 2003).
The substitution of Thr for Ile578 in the spl mutation introduces a site for O-fucosylation into EGF repeat 14 of the N extracellular domain. This
site is fucosylated in SL2 cells and provides a substrate for the further action of Fringe, an enzyme that functions to extend O-fucose glycans. Comparisons of O-fucosylation sites on clotting factors identified
a consensus sequence, C2XXGGS/TC3.
Similar sequences are found in eleven EGF repeats of N, although little is known about which EGF repeats are actually modified in vivo.
However, site-directed mutagenesis of Factor IX and other proteins indicates that Gly residues at the -1 and -2 positions of the consensus are not essential for fucosylation. This raises the possibility that some of the other EGF repeats that contain C2XXXXS/TC3 sequences might be fucosylated. Indeed EGF repeat 25, which contains
C2QNGAS/TC3, is fucosylated by Drosophila SL2 cells and is a substrate for Fringe. SL2 cells fucosylate the sequence C2RNRGTC3 in the spl mutant EGF repeat 14 and the sequence C2LNDGTC3 in wild-type EGF repeat 13. In light of these results, it seems possible that many of the 22 N EGF repeats that contain C2XXXXS/TC3 sequences might be fucosylated.
These include the sequence C2QNEGSC3 in EGF repeat 12, required for Dl to bind and activate N. It is
important to note that the efficiency of O-fucosylation at all these sites is unknown, as well as the efficiency with which O-fucose is extended by Fringe, so that it is possible that even within the same cell individual N molecules may carry different combinations of O-fucose and of extended O-fucose glycans (Li, 2003).
During eye development, fng mutants have little direct effect on R8 specification. In addition, fng is not required for the
spl mutant phenotype. This means that N function during R8
specification is little affected by any extension of O-fucose chains that occurs, unlike N function during wing development. It is possible that O-fucose monosaccharides affect N function during eye development, with or without modification to polysaccharide forms. Consistent with this interpretation, O-fucosylation has been found to be important for many aspects of N function, including others not dependent on Fringe (Li, 2003).
Taken together, these studies suggest that introduction of an
O-fucosylation site into EGF repeat 14 confers sensitivity to Dl on N expressed in R8 precursors, but has little effect on N activity in many other cells. One interpretation is that additional O-fucosylation of N increases sensitivity to ligand, so that N activation occurs in R8 precursors.
The finding that in the wild type R8 cells are insensitive to Dl also suggests another possibility: that EGF repeat 14 has a normal function inhibiting signaling, and that this function is disrupted by O-fucosylation. These two models cannot be distinguished definitively on the basis of current data. The model that EGF repeat 14 has a normal function blocking N signaling in R8 cells is supported by the recessive genetics of the spl mutation, however, because in heterozygous cells that contain wild-type and O-fucosylated EGF repeat 14, the wild-type protein continues to maintain N inactivity in R8 cells. Since EGF repeat 12, which is essential for many aspects of N signaling, contains a potential O-fucosylation site, one very simplistic hypothesis is that whereas O-fucosylated EGF repeats promote N activity, during lateral inhibition EGF repeats lacking
this modification inhibit N activity. It is suggested that during lateral inhibition of neural cells the spatial pattern of N activity is determined by insensitivity of presumptive neural cells to N ligands, and that such insensitivity is regulated by modifications or interactions of EGF repeats on the N extracellular domain (Li, 2003).
Notch and its ligands are modified by a protein O-fucosyltransferase (O-fut1, also known as Neurotic or Ofut1) that attaches fucose to a serine or threonine within EGF domains. By using RNAi to decrease O-fut1 expression in Drosophila, it has been demonstrated that O-linked fucose is positively required for Notch signaling, including both Fringe-dependent and Fringe-independent processes. The requirement for O-fut1 is cell autonomous, in the signal-receiving cell, and upstream of Notch activation. Therefore, O-fut1 activity is required for the cell's ability to receive ligand signals, and would thus be consistent with the hypothesis that the key substrate of O-fut1 is Notch. The transcription of O-fut1 is developmentally regulated, and surprisingly, overexpression of O-fut1 inhibits Notch signaling. Together, these results indicate that O-fut1 is a core component of the Notch pathway, one that is required for the activation of Notch by its ligands, and whose regulation may contribute to the pattern of Notch activation during development (Okajima, 2002).
A mutation has been isolated in the gene encoding O-fucosyltransferase, and analysis of the mutant phenotype confirms the RNAi studies and reveals an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction.
A novel maternal neurogenic gene, neurotic, is essential for Notch signalling. neurotic functions in a cell-autonomous manner, and genetic epistasis tests reveal that Neurotic is required for the activity of the full-length but not an activated form of Notch. neurotic has been shown to be required for Fringe activity. fringe encodes a fucose-specific ß1, 3 N-acetylglucosaminyltransferase that modulates Notch receptor activity. Neurotic is essential for the physical interaction of Notch with its ligand Delta, and for the ability of Fringe to modulate this interaction in Drosophila cultured cells. These results suggest that O-fucosylation catalysed by Neurotic is also involved in the Fringe-independent activities of Notch and may provide a novel on-off mechanism that regulates ligand-receptor interactions (Sasamura, 2003).
Since 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, 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, is not evident in fng mutants. Furthermore, it is thought that Fng does not have a
significant role in lateral inhibition, while it is involved in the generation of
the cell boundary between cells expressing Fng and cells not expressing Fng. Additionally,
an in vitro binding assay revealed that Nti is essential for binding between
Notch and Delta. Based on the previous findings and the present results, it is
proposed that O-glycosylation of Notch EGF repeats has two distinct
roles for binding to Delta. (1) 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. 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. (2)
Addition of GlcNAc to the O-fucose residue by Fng enhances the
interactions between Notch and Delta, modulating the receptor-ligand
interactions. In fact, the expression of fng
shows a high degree of regional specificity, and the boundary of the cells
expressing and not expressing Fng often defines the border of distinct tissue
structures. 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 the 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 (Sasamura, 2003).
To investigate the molecular basis for the requirement for O-linked fucose on Notch, an assay was carried out of the ability of tagged, soluble forms of the Notch extracellular domain to bind to its ligands, Delta and Serrate. Downregulation of O-fut1 by RNAi in Notch-secreting cells inhibits both Delta-Notch and Serrate-Notch binding, demonstrating a requirement for O-linked fucose for efficient binding of Notch to its ligands. Conversely, over-expression of O-fut1 in cultured cells increases Serrate-Notch binding but inhibits DeltaNotch binding. These effects of O-fut1 are consistent with the consequences of O-fut1 overexpression on Notch signaling in vivo. Intriguingly, they are also the opposite of, and are suppressed by, expression of the glycosyltransferase Fringe, which specifically modifies O-linked fucose. Thus, Notch-ligand interactions are dependent upon both the presence and the type of O-fucose glycans (Okajima, 2003).
The requirement for O-Fut1 in Notch signaling has been demonstrated by RNAi in Drosophila
(Okajima, 2002), and by a targeted mutation in the murine Pofut1 gene. One line from a large scale screen for lethal transposable element insertions in
Drosophila has an insertion in the 3' end of O-fut1,
and is predicted to result in replacement of the seven C-terminal amino acids of O-fut1 with
four different amino acids followed by a stop codon. To confirm that this insertion
creates an O-fut1 mutation, animals in which patches of cells were made
homozygous mutant for this allele were examined by mitotic recombination. These animals exhibit classic Notch mutant phenotypes, such as wing notching, thickened wing veins, and loss of sensory bristles on
the notum, consistent with the phenotypes generated by RNAi of O-fut1 (Okajima, 2002). In
developing wing imaginal discs, the expression of targets of Notch signaling, such as Wingless,
is lost in cells mutant for O-fut1. This mutation (referred to hereafter as O-fut1SH) thus
provides an independent demonstration of the requirement for O-fut1 for Notch signaling in
Drosophila, and indicates that the seven C-terminal amino acids of O-fut1 are essential for
function in vivo. The last four amino acids of O-fut1 conform to a consensus signal for
retention in the endoplasmic reticulum, and experiments are in progress to
determine whether the loss of function in O-fut1SH is due to loss of enzymatic activity or to
mislocalization (Okajima, 2003).
The studies presented here indicate that O-fucosylation is required for the physical binding of
Notch to its ligands Dl and Ser. These binding studies are consistent with prior genetic studies,
which positioned a requirement for O-fucosylation in signal receiving cells, upstream of the
cleavages associated with Notch activation (Okajima, 2002). Although the current results do not exclude the
possibility that O-fucose glycans could also act at other steps, and indeed some
influence of O-fut1 RNAi on secretion of Notch extracellular domain fusion proteins is detected, the
requirement for O-fucose for Notch-ligand binding can in principle account for the requirement
for O-fut1 in Notch signaling (Okajima, 2003).
Notably, O-fut1 is required for efficient binding of Notch to both Ser and Dl. This is
consistent with the severe Notch phenotypes observed in vivo when O-fut1 is impaired by
mutation or RNAi. By contrast, elongation of O-fucose by the GlcNAc transferase
Fringe exerts opposing influences on the ability of Notch to bind to Ser and Dl. Fringe has clear and reproducible effects
on both Dl-Notch and Ser-Notch binding. Importantly, these effects of Fringe on Notch-ligand
binding recapitulate its effects on signaling by these two ligands in Drosophila. The ability of
both the O-fucose monosaccharide and elongated forms of O-fucose to influence Notch-ligand
binding, the influence of O-fucosylation on binding by both ligands, and the consistent
correlations between the effects of O-fucosylation on binding in vitro and its effects on signaling
in vivo all argue that O-fucose glycans act at the ligand binding step of Notch signaling (Okajima, 2003).
Beyond their importance to understanding regulation of Notch signaling, these observations thus
provide a striking example of glycosylation as a mechanism for modulating protein-protein
interactions.
With the determination that O-fucosylation affects Notch-ligand binding, attention
must now be turned to elucidating the mechanistic basis for this effect (Okajima, 2003). O-fut1 and Fringe
always act in Notch-expressing cells to influence Notch signaling and Notch-ligand binding: this implicates Notch itself as the relevant substrate. However, the actual sites of glycosylation
on Notch that mediate the effects of these glycosyltransferases remain to be identified. It is also
not yet clear whether the importance of O-fucosylation reflects a role for lectin-like recognition
of Notch by its ligands or other co-factors, or whether instead O-fucose glycans influence Notch-ligand
binding indirectly, by altering the conformation or oligomerization of Notch (Okajima, 2003).
By contrast to the positive requirement for O-fut1 demonstrated by RNAi, over-expression of
O-fut1 enhances Ser-Notch binding but inhibits Dl-Notch binding. It is intriguing that
elevated O-fut1 expression provides a mechanism for differentially modulating the ability of
different Notch ligands to interact with the Notch receptor. Previously, Fringe was the only factor known that
could discriminate between the ability of Delta to activate Notch and that of Serrate to activate
Notch. Indeed, elevated O-fut1 expression might be a mechanism for increasing the
sensitivity of cells to the presence or absence of Fringe. In vivo, Fringe only affects a subset of
Notch signaling events, and it remains unclear why certain processes are sensitive to Fringe
whilst others are insensitive. Although O-fut1 action is the opposite of Fringe, its
effects can be blocked by Fringe; therefore, the relative impact of Fringe on Dl-Notch or Ser-Notch
interactions is expected to be greater in tissues where O-fut1 is expressed at higher levels.
Indeed, even though expression of Fringe alone has no obvious effect on the patterning of notal
bristles, it has a strong effect when O-fut1 is also overexpressed. Overexpression of O-fut1
inhibits Dl-Notch signaling, resulting in the formation of excess sensory bristles, but this effect
is partially inhibited by co-expression with Fringe (Okajima, 2002).
In addition to increasing the sensitivity of Notch signaling events to the presence or
absence of Fringe, elevated O-fut1 expression presents a potential mechanism for modulating
Notch signaling independently of Fringe. Although the in vivo relevance of Notch-ligand
modulation by increased expression of O-fut1 at endogenous levels of expression remains
uncertain, it is noted that certain tissues, such as the lymph gland, express much higher levels of
O-fut1 than surrounding cells (Okajima, 2002). Intriguingly then, in most Drosophila tissues Dl is the sole or
major Notch ligand. However, in the larval lymph gland, a role for Notch signaling in regulating
cell fate decisions during hematopoeisis has recently been described, and Ser, rather than Dl, is
the ligand that regulates Notch in this tissue. These observations provide some support
for the possibility that transcriptional regulation of O-fut1 might provide a mechanism for Notch
pathway regulation (Okajima, 2002), and suggest developmental contexts in which this issue may be investigated further (Okajima, 2003).
Two glycosyltransferases that transfer sugars to EGF domains, OFUT1 and Fringe, regulate Notch signaling. However, sites of O-fucosylation on Notch that influence
Notch activation have not been previously identified. Moreover, the influences of OFUT1 and Fringe on Notch activation can be positive or negative, depending on their levels of
expression and on whether Delta or Serrate is signaling to Notch. This study describes the consequences of eliminating individual, highly conserved sites of O-fucose
attachment to Notch. The results indicate that glycosylation of an EGF domain proposed to be essential for ligand binding, EGF12, is crucial to the inhibition of
Serrate-to-Notch signaling by Fringe. Expression of an EGF12 mutant of Notch (N-EGF12f) allows Notch activation by Serrate even in the presence of Fringe. By contrast,
elimination of three other highly conserved sites of O-fucosylation does not have detectable effects. Binding assays with a soluble Notch extracellular domain fusion
protein and ligand-expressing cells indicates that the NEGF12f mutation can influence Notch activation by preventing Fringe from blocking Notch-Serrate binding. The N-EGF12f
mutant can substitute for endogenous Notch during embryonic neurogenesis, but not at the dorsoventral boundary of the wing. Thus, inhibition of Notch-Serrate binding by
O-fucosylation of EGF12 might be needed in certain contexts to allow efficient Notch signaling (Lei, 2003).
Although the O-fucose site in EGF12 is essential for Fringe inhibition of Serrate signaling in the wing, Fringe still reduces N-EGF12f:AP-Serrate binding. The decrease
in binding is not sufficient to prevent N-EGF12f activation, but there must nonetheless be multiple sites that can contribute to the inhibition of Serrate signaling by Fringe.
There must also be distinct sites that mediate the potentiation of Delta-Notch signaling by Fringe, because N-EGF12f:AP-Delta binding is potentiated almost as effectively as
N:AP-Delta binding. Importantly then, the effects of Fringe on Delta versus Serrate signaling appear to be mediated, at least to some extent, through distinct sites of
O-fucosylation (Lei, 2003).
The importance of additional O-fucose sites is further underscored by the distinct consequences of removal of O-fucose only at EGF12 by the S to A mutation,
compared with removal of O-fucose at all sites by Ofut1 mutation or RNAi. Using a cell aggregation assay, EGF11 and EGF12 of Notch have been shown to have a key
role in ligand binding. Deletion of EGF11 and EGF12 prevents aggregation between Notch-expressing cells and Delta-expressing cells, and a construct including only EGF11 and
EGF12 of Notch is able to confer Delta-binding activity upon cells, albeit with decreased efficiency compared with full-length Notch. Although a role for other EGF repeats in
ligand binding has been suggested based on the consequences of expressing fragments of Notch in the wing imaginal disc, and by cell aggregation experiments with mutant Notch
proteins, EGF11 and EGF12 have generally been considered to be the key EGF domains for ligand binding. However, because RNAi of Ofut1 in S2 cells indicates that
O-fucose is required on Notch for binding to its ligands, yet O-fucosylation of EGF12 is not required for ligand binding, other O-fucosylated EGF domains
must also be required for Notch-ligand interactions. Thus, multiple sites are subject to O-fucosylation, but with different phenotypic consequences (Lei, 2003).
The Drosophila gene neuralized has long been recognized to be essential for the proper execution of a wide variety of processes mediated by the Notch (N) pathway, but its role in the pathway has been elusive. In this report, genetic and biochemical evidence is presented that Neur is a RING-type, E3 ubiquitin ligase. It has been shown that neur is required for proper internalization of Dl in the developing eye, and it has been demonstrated that ectopic Neur targets Dl for internalization and degradation in a RING finger-dependent manner, and that the two exist in a physical complex. Collectively, these data indicate that Neur is a ubiquitin ligase that positively regulates the N pathway by promoting the endocytosis and degradation of Dl (Lai, 2001).
Previous studies have indicated that Dl not only nonautonomously activates the N pathway in neighboring cells, but can also autonomously inhibit the N pathway. For example, a reduction in Dl autonomously potentiates the ability of a cell to receive an N signal, while misexpression of Dl interferes with the ability of a cell to activate the N pathway and can induce N loss-of-function phenotypes. Neur-mediated destabilization of Dl is thus predicted to increase the ability of a cell to receive the N signal, and is therefore consistent with the observed cell-autonomous function of Neur in promoting N pathway activity. The data also suggest a possible explanation for the dominant-negative effect of Dl lacking the intracellular domain, which is predicted to be immune to regulation by Neur (Lai, 2001).
Endogenous neur does not strongly alter Dl level or subcellular localization in the wing disc as assayed by indirect immunofluorescence microscopy, even though neur mutant cells in the eye disc display a clear defect in their ability to internalize Dl. Notably, neur transcripts are not detected in nonsensory organ precursor cells of proneural clusters, even though their requirement for neur can be demonstrated genetically. This suggests that low levels of endogenous Neur may lead to a modification of Dl levels during adult peripheral neurogenesis that is too subtle to observe in the immunofluorescence assay. Neur-mediated destabilization of Dl in the wing imaginal disc may therefore be easily visualized only in gain-of-function experiments where large amounts of Neur are present. Nevertheless, genetic mosaic experiments have convincingly demonstrated that modest changes in the ligand/receptor ratio have a strong influence on cell fate decisions controlled by the N pathway. Thus, Neur need not greatly modify the level of Dl in order to have a significant effect on the activity of the N pathway and the choice of cell fate (Lai, 2001).
Studies of Drosophila dynamin, encoded by shibire (shi), reveal that the activity of the N pathway is particularly dependent upon endocytosis. Shits1 mutants pulsed at the restrictive temperature phenocopy N mutant phenotypes, including neural hyperplasia and thickening of wing veins. Endocytosis and trafficking of Dl and N are abnormal following reduction of dynamin function, and shi function is required in both N signal-sending and -receiving cells, suggesting that both ligand and receptor are regulated by endocytosis. Curiously, misexpression of not only soluble N IC but also membrane-localized full-length N is completely epistatic to shits, indicating that endocytosis is not essential for signal transduction downstream of the N receptor. The requirement for shi in N signal-receiving cells might be simply explained if it functions in Neur-regulated endocytosis of Dl. In this case, biasing the ligand/receptor ratio by misexpression of full-length N would be sufficient to bypass the requirement for dynamin. Consistent with this, preliminary experiments indicate that the ability of Neur to downregulate Dl is compromised when dynamin function is reduced (Lai, 2001).
It is also emphasized that multiple mechanisms must exist for internalization of Dl, since endocytosis of Dl still occurs in wing and eye disc neur clones, and Dl accumulates in large intracellular apical vesicles in the presence of dominant-negative NeurDeltaRF. In addition, Dl localizes to vesicles in a dynamin-independent fashion in the pupal wing. It is suggested that ubiquitination of Dl by Neur represents a mechanism for regulated endocytosis and subsequent degradation of Dl, but additional means for clearance of Dl from the plasma membrane must exist, possibly including constitutive membrane recycling or pinocytosis (Lai, 2001).
neur is essential for many, but not all, lateral inhibitory and inductive processes mediated by N. For example, neur is absolutely required for multiple steps during PNS development and for lateral inhibition of photoreceptors, but is dispensable for processes such as lateral inhibition of wing veins and induction of wing margin. Nevertheless, ectopic Neur and NeurDeltaRF could interfere with all of these processes, consistent with the proposed function of Neur in regulating Dl, a 'core' N pathway component involved in all of these processes. In light of the findings presented here, attempts were made to identify common features of Neur-dependent Dl-mediated processes (Lai, 2001).
It has been previously observed that Dl and N expression are coincident in some settings and complementary in others. Notably, Dl and N expression are coincident or overlapping in most settings that require Neur, including in proneural clusters of the imaginal discs and the pupal notum, and in the developing eye imaginal disc. Conversely, Dl and N are complementary or highly asymmetric in Neur-independent developmental settings such as disc and pupal wing vein development, and at the wing margin. An attractive hypothesis is that Neur functions to bias the relative levels of N and Dl in settings where both ligand and receptor are coexpressed on a cell-by-cell basis; in other settings where ligand and receptor expression are highly asymmetric or exclusive, Neur may not be required (Lai, 2001).
Activation of the Notch (N) receptor involves an intracellular proteolytic step triggered by shedding of the extracellular N domain (N-EC) upon ligand interaction. The ligand Dl has been proposed to effect this N-EC shedding by transendocytosing the latter into the signal-emitting cell. Dl endocytosis and N signaling are greatly stimulated by expression of neuralized. neur inactivation suppresses Dl endocytosis, while its overexpression enhances Dl endocytosis and Notch-dependent signaling. neur encodes an intracellular peripheral membrane protein. Its C-terminal RING domain is necessary for Dl accumulation in endosomes, but may be dispensable for Dl signaling. The potent modulatory effect of Neur on Dl activity makes Neur a candidate for establishing signaling asymmetries within cellular equivalence groups (Pavlopoulos, 2001).
Static pictures of Dl localization do not allow an unambiguous conclusion of whether intracellular Dl is endocytosed or blocked in its secretory trafficking. The former hypothesis is favored for three reasons: (1) intracellular Dl often colocalizes with endocytosed fluorescent dextran; (2) if Dl were retained in the endoplasmic reticulum or Golgi, it would not be available at the cell surface where signaling is taking place, yet, concomitant with increased endocytosis, Neur is able to stimulate Dl signaling and (3) wt Neur protein is found mostly at the plasma membrane, so it is more likely to affect endocytic events rather than steps in secretory processes (Pavlopoulos, 2001).
The nonautonomous effect of neur- clones on lateral inhibition favors a role for Neur in signal-emitting, rather than signal-receiving, cells. Such a function is consistent with the fact that Neur is an intracellular peripheral membrane protein expressed preferentially in the signal-emitting cells during lateral inhibition, such as the neuroblasts, SOPs, and central provein cells. In agreement with a role for Neur in generating the Dl signal, epistasis analyses have shown that neur is required to express the embryonic neural suppression ('antineurogenic') phenotype associated with ligand-dependent N gain-of-function (gof) mutants. neur is dispensable for the constitutive activity of ligand-independent N variants. Interestingly, some N variants that are Dl independent are also shi (Dynamin) independent. Taken together, these data point to the involvement of Neur and Dynamin in processes upstream of (or parallel to) N activation by Dl. The implication of Neur in endocytic regulation suggests an important role for endocytosis in events leading up to N activation (Pavlopoulos, 2001).
If Dl endocytosis and Dl-N signaling are causally linked, then this analysis of the NeurDeltaRING-GFP mutant poses a paradox: although NeurDeltaRING-GFP does not detectably stimulate Dl endocytic trafficking (or turnover), it retains the ability to enhance Dl signaling. This could mean that the above model is wrong and endocytosis is simply a consequence of Dl-N stimulation, rather than a prerequisite for Dl signaling. Alternatively, the absence of detectable Dl internalization upon coexpression of NeurDeltaRING-GFP does not necessarily preclude the possibility that early endocytic events (e.g., recruitment of Dl into coated pits) that are undetectable by light microscopy are initiated by NeurDeltaRING-GFP. Such events might be sufficient to stimulate ligand-dependent N cleavage and activation. Ultrastructural analysis will be required to distinguish between these alternative models (Pavlopoulos, 2001).
Removal of the Neur RING domain does seem to adversely affect its ability to stimulate N signaling in some contexts: UAS-neurDeltaRING yields phenotypes indicative of a negative effect on N signaling (tufted bristles, thick veins, and notched wings) with most Gal4 driver lines, although in certain cases, positive effects are also observed (shaft to socket transformation). Context-dependent variability with the UAS-neurDeltaRING-GFP construct suggests that these differences do not result from the presence of the GFP moiety but rather from the type of assay employed. In fact, NeurDeltaRING-GFP coexpressed with Dl blocks N signaling within the omb-Gal4 domain, where wt Neur and Dl are able to induce Wg, even though the nonautonomous signaling (at the borders of the omb-Gal4 domain or at the borders of FLP-out clones) appears unaffected by the RING deletion. It is possible then that NeurDeltaRING can exert negative effects on Dl-N signaling in a cell-autonomous fashion and positive effects in a cell-non-autonomous fashion. The cell-autonomous block in N signaling could be due to the block in Dl turnover and its accumulation at the apical membrane, because it has been proposed that high levels of Dl may sequester N receptor molecules in unproductive cis complexes (Pavlopoulos, 2001).
Two major models for Dl signaling have been put forward. In one, the active Dl species is proposed to be the extracellularly cleaved, secreted Dl-EC fragment, because it is produced by the metalloprotease Kuzbanian (Kuz), and the kuz lof phenotype is similar to the N lof phenotype. In the other, binding of cell surface-tethered Dl to N on the apposing cell has a dual impact: activating extracellular cleavage of Notch and mediating the transendocytosis into the signal-sending cell of N-EC complexed with Dl. The observations in this paper suggest that Neur could act intracellularly in the signal-sending cell to promote assembly of a productive Dl-N complex and to trigger its endocytosis. Concomitantly with endocytosis, Neur leads to a drastic reduction in the levels of the Dl-EC fragment, even as Dl-N signaling is increased. It therefore appears unlikely that Dl-EC is the active signal that stimulates N in the wing disk. This leaves unanswered at present the question of why Kuz is needed for N signaling. Perhaps Kuz has pleiotropic activity and acts on some other protein(s) required for N activation, and Kuz-dependent Dl cleavage is a secondary effect. Better characterization of the different Dl isoforms, including their localization and trafficking, will be required to understand the detailed mechanism of Dl-N activation (Pavlopoulos, 2001).
Despite the proposed role of Neur to promote Dl signaling, it is also noted that Dl can signal in the absence of Neur, inasmuch as there are instances of Dl signaling where Neur is not detectably expressed, such as from the germline to ovarian follicle cells. N target gene expression is indeed induced by Dl in the absence of neur. With the caveat that available detection methods may fail to detect low levels of neur expression, it is proposed that two types of Dl signaling may exist: basal signaling that does not require Neur activity and high-intensity signaling that does. During neurogenesis, basal Dl-N signaling probably takes place during early stages among all cells within proneural clusters, where Dl and N are uniformly expressed but Neur is absent. Upon expression of neur by a nascent neural precursor, signaling becomes asymmetric, since the neighboring cells receive more intense signal even though Dl and N levels have not changed. The absolute requirement for neur in neurogenesis suggests that basal 'mutual' inhibition is insufficient to permanently block proneural protein activity. Indeed, the E(spl) bHLH Notch targets, which are the main antagonists of proneural proteins, are not expressed in neur- embryos or clones, suggesting that their expression may be induced only by intense Neur-dependent 'lateral' inhibitory signaling (Pavlopoulos, 2001).
This hypothesis can be extended to propose that Neur may be required more stringently in instances in which a novel asymmetry has to be imposed upon uniform basal N-Dl signaling. neur is not required at the wing DV boundary, where asymmetry is imposed by Fringe or in the egg chamber, where asymmetry is imposed by expression of N and Dl in distinct cells. Similarly, neur is not essential during lateral inhibition within the provein. Despite its expression there and its dramatic effect on Dl localization, neur lof clones yield normal looking veins with only minor thickenings. It is believed that neur is not crucial for this process because wing patterning mechanisms place N and Dl in different cells: Dl expression is most intense within the central proveins and N expression is most intense within the lateral proveins (Pavlopoulos, 2001).
Notch signaling regulates cell fate decisions during development through local cell interactions. Signaling is triggered by the interaction of
the Notch receptor with its transmembrane ligands expressed on adjacent cells. Recent studies suggest that Delta is cleaved to release an
extracellular fragment, DlEC, by a mechanism that involves the activity of the metalloprotease Kuzbanian; however, the functional
significance of that cleavage remains controversial. Using independent functional assays in vitro and in vivo, the biological
activity of purified soluble Delta forms were examined; it is concluded that Delta cleavage is an important down-regulating event in Notch signaling. The
data support a model whereby Delta inactivation is essential for providing the critical ligand/receptor expression differential between neighboring cells in order to distinguish the signaling versus the receiving partner (Mishra-Gorur, 2002).
Western analysis of extracts from tissues and cultured Drosophila S2 cells show that the ligand Delta is cleaved at an extracellular site close to the transmembrane domain, shedding a fragment that encompasses most of the extracellular domain (DlEC). Conditioned medium from S2 cells stably expressing Delta (S2-Dl) was used to purify DlEC to homogeneity by affinity chromatography using the 9B monoclonal antibody (C594.9B). Resolution of the highly purified product by SDS-PAGE and silver staining demonstrates two species migrating as a doublet of 63 and 65 kD, with the 63 kD species being the predominant form. NH2-terminal sequence analysis revealed a single sequence consistent with the putative NH2 terminus resulting from the signal peptide cleavage. Direct chemical COOH-terminal sequence analysis determined that the COOH-terminal residue of both isoforms is alanine. These results were corroborated by tryptic digestion followed by mass spectrometry, which revealed the existence of two peptides ending in the sequence LTNA and ... QYGA. It is concluded that Delta is cleaved at two distinct sites: COOH-terminal to Ala581 and Ala593, respectively. Henceforth, these two isoforms are referred to as DlEC581 and DlEC593 (Mishra-Gorur, 2002).
In an attempt to explore the functional significance of the two extracellular cleavages in Delta, truncated soluble molecules mimicking the DlEC581 and DlEC593 were generated. In addition, the Ala581 and Ala593 amino acids were mutagenized to serine (henceforth Ala581Ser and Ala593Ser). The constructs were transfected into Drosophila S2 cells, which endogenously express Kuzbanian but not Delta. Transfection of the DlEC581 and DlEC593 constructs effectively generated soluble secreted products, with DlEC593 exhibiting a slightly different molecular mass, consistent with the 11-amino-acid difference in their COOH-termini. When expressed in S2 cells, both the Ala581Ser and Ala593Ser mutants were cleaved to generate a product of essentially the same size as DlEC. In addition, an Ala581,593Ser double mutant was generated which was also cleaved to generate a product similar to DlEC. The cis or trans requirement of Kuz in Delta cleavage was assessed using the S2 cell-culture system. S2 cells stably expressing either WT Kuz or dominant-negative Kuz, when mixed with S2-Dl cells, do not affect Delta cleavage; Kuz effects are seen only when it is cotransfected with Delta into the same cell (Mishra-Gorur, 2002).
It has been established that cells expressing Notch aggregate with Delta-expressing cells. Although a rigorous, in vivo demonstration that this interaction is direct is still lacking, it is known that specific regions in the extracellular domain of Notch and Delta are necessary and sufficient for aggregation. Attempts were made to examine whether WT DlEC, DlEC581, and DlEC593 interact with Notch and thus inhibit the normal Notch-Delta-mediated cell aggregation (Mishra-Gorur, 2002).
Preincubation of S2-N cells with concentrated conditioned medium from S2-Dl cells causes a >60% inhibition of aggregation rate. In contrast, concentrated conditioned medium from S2 cells stably expressing each of the mutant forms of soluble Delta show essentially no inhibitory effect in the aggregation assay (Mishra-Gorur, 2002).
The medium from S2-Dl-expressing cells was fractionated on an anti-Delta (9B) antibody affinity column, and selected fractions were tested for their inhibitory effect in the aggregation assay. All the inhibitory activity in the flowthrough of the affinity column. Further, the purified DlEC fractions shows essentially no inhibitory activity. A mild inhibition in aggregation (<20%) was seen at concentrations of DlEC >0.5 µM, indicating that DlEC has a very weak affinity for Notch and is not an effective competitive inhibitor of Notch-Delta aggregation. Western blot analysis of the different fractions during purification of DlEC showed that the flow through contains full-length Delta, suggesting that the inhibitory effect could be either attributed to this Delta protein species or to an unknown activity which copurified with it. In any case, it is noted that these experiments reveal the existence of a Notch agonist activity, other than DlEC, in the supernatant of the S2-Dl cells (Mishra-Gorur, 2002).
The S2-Dl-derived inhibitory activity was further examined by size exclusion chromatography in neutral aqueous buffer to avoid the harsh elution conditions of the affinity column (i.e., pH 2.8). When S2-Dl conditioned medium was fractionated on a Sephadex-200 HR FPLC size exclusion column, all of the inhibitory activity was seen to elute in the void volume of the column (Mr>600 kD). Western blot analysis also demonstrated that this fraction was devoid of DlEC, which eluted in subsequent fractions. It is important to note that a band corresponding in size with full-length Delta is seen in the void-volume fractions. These data corroborate the notion that the DlEC fragment does not compete with the Notch-Delta interaction mediating the cell aggregation (Mishra-Gorur, 2002).
This analysis was extended by examining the activity of the soluble Delta molecules using independent in vitro assays of Notch activation. All of the in vitro assays employed consistently indicate that the soluble forms of Delta are not active, and support the notion that cleavage of Delta corresponds to an inactivation of the ligand. However, they also corroborate the existence of a soluble agonist activity in fractions containing small amounts of full length Delta (Mishra-Gorur, 2002).
The in vitro studies were extended by an assessment of the activity of the mutant constructs in transgenic flies. Flies carrying the various Delta mutants were generated and the effects of expression of the different DlEC isoforms and Delta mutants were analyzed in vivo. Expression was driven by the eye specific glass (pGMR) promoter, which is active in all cells posterior to the morphogenetic furrow. Flies expressing DlEC581 and DlEC593 exhibited mild eye phenotypes. The underlying mechanism of the weak effects associated with soluble ligand expression are not understood; however, the severity of phenotypes of the various transgenic lines varies from mild to no phenotype, suggesting a link with the level of over expression. This may correlate with the slight inhibition of aggregation observed with micromolar amounts of purified DlEC in the in vitro aggregation assay (Mishra-Gorur, 2002).
In contrast, a severe glassy eye phenotype is exhibited with the Ala581Ser and Ala593Ser mutants. Significantly, very similar results are seen with pGMR driven overexpression of WT Delta, consistent with the notion that the cleavage site mutations, in addition to being ineffective at preventing cleavage, do not significantly alter the biological activity of Delta. The in vivo activity of the mutant Delta molecules corroborates the results of the cell-based analysis. It is important to note that unlike the strong phenotypes associated with the overexpression of the WT ligand, or the full-length mutant ligands that are normally cleaved in the aforementioned cell based assays, the soluble forms have mild effects. The expression of the soluble Delta isoforms in every context examined could at best elicit only mild phenotypes consistent with the notion that these molecules are inactive (Mishra-Gorur, 2002).
The finding that the proteolytic processing of Delta releases soluble DlEC raised the obvious question of the functional significance of this cleavage. Even though several studies have addressed this question, either directly or indirectly, in flies, nematodes and vertebrates, it is unclear whether this is an antagonistic or agonistic event in Notch signaling. The initial characterization of soluble fractions of Delta suggested an agonistic function for the DlEC. More rigorous biochemical characterization presented here clearly shows Delta proteolysis yields more than one DlEC (DlEC581 and DlEC593), neither of which exhibit significant biological activity. Furthermore, the previously reported soluble activity is most likely attributed to trace levels of full-length Delta in the cell culture media. It is concluded is that the proteolytic processing of Delta is a step that renders this Notch ligand inactive (Mishra-Gorur, 2002).
Previous studies demonstrated a central role for Kuzbanian in Delta processing both in cell-based assays and in vivo by mutant analysis. The existence of two DlEC products (DlEC581/593) indicates more than one cleavage event occurs in the extracellular domain of Delta. It is important to note that the DlEC581 product is far more abundant as compared to DlEC593 and experiments using KuzDN result mainly in the reduction of the 581 form. Whether or not Kuzbanian alone or additional enzyme activity is responsible for these cleavages requires further investigation. Regardless of the mechanism of cleavage, both of the Delta products have proven to be biologically inactive. Therefore, it is reasonable to conclude that processing in general results in ligand inactivation (Mishra-Gorur, 2002).
Based on these results, it is suggested that the agonistic activity, previously reported to be associated with the medium from Delta expressing cells, is not due to the activity of DlEC. However, it is noted that the present study detected the presence of a 'soluble' activity in the medium, raising the possibility that such an activity may after all exist in vivo. Formally at least, this activity can be attributed to the detectable quantities of full length Delta in the medium or to another yet-to-be-determined molecule. It is not inconceivable that soluble, full-length, membrane-associated Delta may in fact be secreted into the medium even if only to act on a neighbor rather than over long distances. For instance, in the case of Wingless, the existence of membrane exovesicles as a vehicle for Wingless delivery has been documented. Whether a soluble, biologically significant Delta activity can be generated by exocytic events remains to be tested, but this is worth considering (Mishra-Gorur, 2002).
Despite the uncertainty of the role of ligand processing, several studies have attempted to use soluble forms of the ligand as an agonist of the receptor with variable success. However, a common element in these studies is that the soluble forms display activity only if they are forced into an oligomeric state either via Fc fusions or by immobilization on a matrix. The biologically inactive DlEC fragment secreted in the medium does not have a natural tendency to oligimerize because it exists in a monomeric state (as judged by gel filtration and centrifugal/sedimentation studies. Furthermore, the inactivity of DlEC expressed in vivo indicates that a biologically relevant mechanism for immobilization of the DlEC so as to make it active is nonexistent. Therefore, it is of utmost importance to consider the physiological relevance of continued attempts to employ soluble ligands as Notch agonists (Mishra-Gorur, 2002).
Irrespective of the potential requirement for oligomerization or immobilization as an essential activation step for the ligand, it has been proposed that endocytosis of the dissociated Notch extracellular domain bound to Delta into the Delta-expressing cells (transendocytosis) is a critical part of the Notch signaling mechanism. If such a mechanism is essential for Notch activation, then the blocking of an endocytic event may result in inhibition of signaling. This notion is also compatible with the in vivo analysis that demonstrates that membrane-tethered forms of either Delta or Serrate lacking the intracellular domain cannot undergo effective endocytosis, and hence behave as antagonists of Notch signaling. However, the present analysis shows that Delta molecules fixed on the cells, similar to a molecule immobilized on a matrix, is still capable of activating the Notch receptor. This observation would then favor the hypothesis that endocytosis of Delta may be a facilitating but not necessarily an essential part of Notch signaling (Mishra-Gorur, 2002).
In assessing the developmental significance of Delta cleavage, the activity of Kuzbanian needs to be examined more closely. Although the initial link between Notch signaling and Kuzbanian was reported to involve Notch processing, genetic data show that multiple copies of Delta can suppress the phenotypes associated with dominant-negative Kuzbanian (KuzDN) expression. This observation is compatible with the notion that Delta cleavage produces an active soluble ligand. However the mechanism of action of KuzDN is not known and it may be equally plausible to consider that KuzDN acts by sequestering Delta, such that the addition of more WT Delta molecules suppress the KuzDN phenotype. It is also worth emphasizing that whereas the dominant-negative forms of Kuzbanian inhibit Delta cleavage, and that Delta cleavage products are not detected in loss of function kuz embryos, it is quite possible that the Kuzbanian-Delta interaction is indirect. The original proposal that Kuzbanian is involved in the proteolytic processing of Notch has been challenged by subsequent experimentation. Indeed, recent reports documenting Kuzbanian cleavage of Notch rely on deletion mutants of the receptor that are susceptible to cleavage, bringing further uncertainty to the physiological relevance of Kuzbanian acting on Notch directly (Mishra-Gorur, 2002).
A model to explain the role of Delta down-regulation by proteolysis must consider the mechanism of action of the Notch ligands. Delta can influence Notch through two modes of action: in trans, where Notch and Delta are presented on adjacent cells and Delta can act as agonist, or in cis, where Notch and Delta are presented on the same cell and Delta (and Serrate) can act as a dominant-negative antagonist. It is well established that cells in tissues undergoing Notch signaling can express Notch and Delta simultaneously. For instance, in the early Drosophila embryo, all cells in the proneural clusters, the group of cells which will eventually segregate into epidermal and neuronal lineages via Notch-Delta signaling, express both Notch and Delta. However, in order for proper signaling to occur, there must be a distinction between a signaling versus a receiving cell. The accumulated studies to date suggest that the critical parameter for a cell to be a receiving or signaling cell is the ratio rather than the absolute expression levels of Delta and Notch. Moreover, feedback loops may be responsible for consolidating and amplifying a given state (ratio). Thus, a mechanism that inactivates Delta in a given cell may contribute to the feedbacks that are necessary to establish a critical expression differential between two neighbors (Mishra-Gorur, 2002).
Mosaic analysis in Drosophila during cell fate acquisition in the neuroectoderm has demonstrated that Kuz is required in cells to receive signals that inhibit the neural fate. These signals are known to be transmitted through the Notch receptor and this cell autonomous effect of Kuz is consistent with studies in nematodes. Similarly, using a cell-culture system, it has been found that dominant-negative or WT forms of Kuzbanian can affect Delta only when cotransfected in the same cell, and have no effect when transfected into adjacent cells. Hence, it is suggested that Kuz acts on Delta in the same cell, although it is not clear whether the cleavage occurs at the cell surface or inside the cell. In either case, it is proposed that this proteolysis renders Delta incapable of interacting with Notch either on an adjacent cell or on the same cell (Mishra-Gorur, 2002).
Therefore, a model is favored whereby proteolytic processing of Delta on a Notch/Delta-expressing cell has the overall effect of rendering that cell the signal receiving cell by (1) alleviating the dominant-negative activity of Delta toward Notch on that cell, and (2) down-regulating the Delta available to signal Notch on adjacent cells. Consistent with this model, a similar role has been proposed for the Neuralized ubiquitin ligase in Delta down-regulation. Interestingly, because Neuralized is not required in all Notch dependent developmental contexts, it has been emphasized that multiple mechanisms must exist to clear Delta from the plasma membrane. The above model is also compatible with the possibility that Kuzbanian may play more than one role in Notch signaling. For example, if Kuz is somehow involved in the activation of the Notch receptor, the existence of an activity such as Kuzbanian in a particular cell, which is able to simultaneously enhance receptor function and inactivate the ligand, is a hypothesis worth testing. One of the predictions of the proposed scheme is that Kuzbanian activity must be differentially regulated between critical neighbors. More experimentation will be necessary to confirm or discount this hypothesis and indeed this model (Mishra-Gorur, 2002).
In Drosophila, Notch signaling regulates binary fate decisions at each asymmetric division in sensory organ lineages. Following division of the sensory organ precursor cell (pI), Notch is activated in one daughter cell (pIIa) and inhibited in the other (pIIb). The E3 ubiquitin ligase Neuralized localizes asymmetrically in the dividing pI cell and unequally segregates into the pIIb cell, like the Notch inhibitor Numb. Furthermore, Neuralized upregulates endocytosis of the Notch ligand Delta in the pIIb cell and acts in the pIIb cell to promote activation of Notch in the pIIa cell. Thus, Neuralized is a conserved regulator of Notch signaling that acts as a cell fate determinant. Polarization of the pI cell directs the unequal segregation of both Neuralized and Numb. It is proposed that coordinated upregulation of ligand activity by Neuralized and inhibition of receptor activity by Numb results in a robust bias in Notch signaling (Le Borgne, 2003).
Recent studies have indicated that endocytosis of Dl is critical for N activation. (1) Dynamin-dependent endocytosis is not only required for signal transduction but is also required in signal-sending cells to promote N activation. (2) Endocytosis-defective Dl proteins have reduced signaling capacity. (3) The E3-ubiquitin ligases Neuralized (Neur) in Drosophila and Mind bomb (Mib) in zebrafish promote endocytosis of Dl and appear to be required for efficient activation of N by Dl. It has been proposed that Dl endocytosis facilitates the S2 cleavage of N at the surface of the signal-receiving cell. Neur is unequally segregated during asymmetric division of the pI cell, upregulates endocytosis of Dl in the pIIb cell, and plays a critical role in generating cell fate diversity. It is proposed that Neur acts as a cell fate determinant during asymmetric cell divisions (Le Borgne, 2003).
To examine whether asymmetry in N ligands distribution may play a role in generating cell fate diversity during asymmetric divisions, the subcellular distribution of Dl and Ser was studied in the sensory organ lineage. In mitotic pI cells, Dl and Ser are uniformly distributed around the cell cortex and are equally partitioned into both daughter cells. In both pI daughter cells, Dl and Ser accumulated at the apical cell cortex as well as in intracellular dots of 0.5 ± 0.2 μm in diameter. These dots are coated by Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate). Hrs binds ubiquitinated proteins via its ubiquitin-interacting motif and sorts endocytic cargos into the lumen of multivesicular bodies (MVBs). Therefore, these Dl-positive vesicles appear to be large endocytic vesicles that probably correspond to MVBs. These Dl-positive vesicles also contain Notch extracellular domain (NECD) and NICD epitopes. Strikingly, a higher number of large Dl-positive vesicles were seen in the anterior signal-sending pIIb cell (5.0 ± 2.2) than in the posterior signal-receiving pIIa cell (2.0 ± 1.5). This asymmetry in Dl endocytosis is established independently of the unequal partitioning of Numb. Indeed, anterior pI daughter cells are shown to accumulate a higher number of Dl-positive vesicles than posterior pI daughter cells in numb2 and numb15 mutant clones. Thus, asymmetry in Dl endocytosis does not depend on Numb (Le Borgne, 2003).
Recent studies have suggested that endocytosis of Dl is promoted by the ubiquitination of Dl by Neur, a RING finger-type E3-ubiquitin ligase required for N signaling. Neur is found in a complex with Dl and is required for Dl ubiquitination. Finally, Neur stimulates the accumulation of Dl into intracellular vesicles in imaginal disc cells. The latter conclusion was, however, based on the analysis of steady-state levels of Dl, making it difficult to unambiguously conclude whether Neur promotes Dl endocytosis or favors direct sorting from the Golgi to intracellular vesicles. To discriminate between these two possibilities and to test whether Neur regulates Dl trafficking in sensory cells, an ex vivo assay was developed for endocytosis. Internalization of Dl was followed in living epithelial cells using antibodies recognizing the extracellular part of Dl. Briefly, the single-layered epithelium corresponding to the pupal notum was dissected and cultured in the presence of anti-Dl antibodies. Following medium changes and fixation, the uptake of anti-Dl antibodies was revealed using secondary antibodies. Anti-Dl antibodies were found to be specifically internalized in the pIIa and pIIb cells. Internalized anti-Dl antibodies colocalize with Dl into large Dl-positive vesicles. Internalization of anti-Dl requires dynamin activity and is not observed at 4°C. Together, these results indicate that anti-Dl interacts with Dl at the cell surface and that Dl-anti-Dl complexes are endocytosed in sensory cells (Le Borgne, 2003).
This assay was used to examine the function of neur. Clones of neur1F65 mutant cells have been shown to exhibit a neurogenic phenotype with too many pI cells being specified. The progeny of these mutant pI cells produce no external sensory structures indicating that pIIa cells have been transformed into pIIb-like cells. These cell fate transformations are associated with defects in Dl trafficking. High levels of anti-Dl remain at the surface of neur1F65 mutant cells and internalization of anti-Dl is drastically reduced. It is concluded that neur is required for the endocytosis of Dl in sensory cells (Le Borgne, 2003).
This defect in Dl endocytosis was quantified on fixed tissues. neur mutant pI cells and pIIb-like progeny cells were found to accumulate high levels of Dl at the cell surface. Accumulation of Dl at the cell surface is consistent with the proposed function of Neur in the internalization and degradation of Dl. Quantification of Dl-positive vesicles in neur mutant clones revealed that mutant pIIb-like cells contain much fewer Dl-positive vesicles than wild-type pIIb cells. Thus, in the absence of neur function, both pI daughter cells have the same reduced number of Dl-positive vesicles. Furthermore, a similar distribution of Dl-containing vesicles is seen in the wild-type pIIa cells, which do not inherit Neur, and in the neur mutant pIIb-like cells. These comparisons indicate that neur is required to upregulate the endocytosis of Dl in the pIIb cell (Le Borgne, 2003).
Upregulation of Dl endocytosis in the pIIb cell may result from higher levels of Neur in this cell. To test this hypothesis, the localization of Neur was examined. The Neur protein is detectable in the pI cell and in its progeny cells, but not in epidermal cells. Neur is perinuclear in prophase and localized asymmetrically at the anterior cortex during prometaphase. At telophase, Neur specifically segregates into the anterior daughter cell. At cytokinesis, Neur uniformly redistributes at the cortex and in the cytoplasm in the pIIb cell. Localization of Neur at mitosis is identical to the one described for Partner of Numb (Pon). Consistently, Neur colocalizes with Pon-GFP throughout mitosis. Asymmetric localization of Neur is also seen in the pIIb and pIIa dividing cells. Specificity of anti-Neur antibodies was demonstrated by absence of staining in neur mutant pI cells. Unequal segregation of Neur does not depend on numb activity. Conversely, unequal segregation of Numb does not depend on neur activity. Thus, the numb-independent unequal segregation of Neur into the pIIb cell provides a simple explanation for the upregulation of Dl endocytosis in the pIIb cell (Le Borgne, 2003).
To test the functional significance of Neur unequal segregation, Neur was overexpressed in pI cells. Overexpression of Neur using neurP72GAL4 fails to affect the unequal partitioning of Neur at pI mitosis and the pIIa/pIIb decision but instead results in a weak double-socket phenotype associated with a shaft-to-socket transformation. This fate transformation is known to result from high levels of Delta-Notch signaling and is opposite that of the socket-to-shaft transformation seen in neur mutant clones. Moreover, this shaft-to-socket transformation may result from the equal partitioning of Neur (but not Numb) in the two pIIa daughter cells which can also be observed at low frequency. Thus, these observations support the notion that unequal segregation of Neur is functionally important (Le Borgne, 2003).
The mechanisms by which Neur localized at the anterior cortex of the dividing pI cell were investigated. The role of the cytoskeleton was studied by applying drugs to cultured nota. Colcemid, a microtubule-depolymerizing agent, was found to have no significant effect. In contrast, both Latrunculin A, an agent that depolymerizes actin microfilaments, and the myosin motor inhibitor butanedione-2-monoxime (BDM) strongly impaired or completely inhibited the asymmetric localization of Neur. Thus, both myosin motor activity and an intact actin cytoskeleton are required for the formation and/or maintenance of the Neur crescent at the anterior cortex of the dividing pI cell. These requirements for Neur localization are similar to the ones seen earlier for Numb and Pon. Neur also behaves in a manner similar to Numb and Pon in that localization of Neur at the anterior cortex of the pI cell depends on planar polarity genes and on the polarity genes discs-large and pins. Moreover, mispartitioning of Neur in dlg and pins mutant cells correlates with a loss in asymmetric internalization of Dl. These data indicate that Neur and Numb share part of the same molecular machinery to localize asymmetrically in the pI cell (Le Borgne, 2003).
Unequal segregation of Neur in the anterior pIIb cell suggests that Neur acts in this cell to promote adoption of the pIIa fate by the posterior cell. To test whether neur activity is indeed required in the pIIb cell, clones within the sensory organ lineage were generated. Mitotic recombination in the pI cell produces one neur mutant cell and one wild-type cell. Importantly, the anterior daughter cell inherits Neur, regardless of its genotype. Thus, when the anterior cell is neur mutant, the posterior cell is predicted to adopt a pIIa fate whatever the requirement for neur activity. However, two different outcomes are predicted when the posterior cell is mutant. If neur activity is required in the signal-receiving cell, the posterior cell is predicted to adopt a pIIb-like fate activity. This should result in a bristle loss phenotype. In contrast, if neur acts in the signal-sending cell, the mutant posterior cell is predicted to become a pIIa cell. This mutant pIIa cell should then produce two mutant cells unable to signal, hence leading to bristle duplication. Mitotic recombination induced at 0-6 hr before puparium formation (PF), when most macrochaete pI cells are specified but have not yet divided, produces flies with double-shaft bristles on the head, thorax and at the wing margin. No macrochaete loss was detectable. This double-shaft phenotype appears to result from wild-type pIIb/mutant pIIa pairs because sensory organs composed of two mutant shaft cells and wild-type pIIb progeny cells were detected at 20 hr after PF. Reciprocally, a sheath-to-neuron transformation was observed in mutant pIIb/wild-type pIIa pairs. These data show that neur is required for the socket/shaft and neuron/sheath fate decisions and further indicate that neur acts in the pIIb cell to specify the pIIa cell (Le Borgne, 2003).
The Notch intercellular signalling pathway is important throughout development, and its components are modulated by a variety of cellular and molecular mechanisms. Ligand and
receptor trafficking are tightly controlled, although context-specific regulation of this is incompletely understood. During sense organ precursor specification in Drosophila,
the cell adhesion molecule Echinoid colocalises extensively with the Notch ligand, Delta, at the cell membrane and in early endosomes. Echinoid facilitates efficient Notch
pathway signalling. Cultured cell experiments suggest that Echinoid is associated with the cis-endocytosis of Delta, and is therefore linked to the signalling events that have
been shown to require such Delta trafficking. Consistent with this, overexpression of Echinoid protein causes a reduction in Delta level at the membrane and in endosomes. In
vivo and cell culture studies suggest that homophilic interaction of Echinoid on adjacent cells is necessary for its function (Rawlins, 2003).
Therefore, both in vivo and in culture Ed protein is strongly associated with Dl at the cell membrane and in the early endosome compartment. Several lines of evidence suggest
that Ed self associates in trans. Ed expression promotes the adhesion of cultured cells, while genetic clonal analysis shows that in vivo Ed protein cannot accumulate at the
cell membrane if it is absent from the adjacent cell. Moreover, this genetic analysis suggested that such a trans interaction might be important for function (Rawlins, 2003).
Ed is not essential for Notch signalling but has a modulatory effect. The basis of this effect must be relatively subtle, since no strongly visible difference is found in
expression pattern, level, or subcellular localization of Dl, N or E(spl) in ed mutant clones. The idea is favored that Ed influences PNC resolution as part of the
specific process that drives the singling out of individual SOPs. In other words, it is a part of a 'symmetry breaking' apparatus. There are two lines of evidence to suggest
that Ed functions to inhibit the transition from PNC cell to SOP. (1) No more than four SOPs are selected from each PNC even in null ed alleles. (2) ed interacts
particularly strongly with ase, which is expressed on the transition from PNC to SOP. It is suggested that the role of ed is analogous to that proposed for
sca. Based on analysis in the eye, it is envisaged that singling out causes several cells to begin to become resistant to Dl ('pre-SOPs'), but a specific genetic
mechanism involving sca and gp150, encoding a leucine-rich repeat (LRR) protein that is required for viability, fertility and proper development of the eye, wing
and sensory organs, causes all but one of these unwanted SOPs to revert and once again become responsive to Dl from the selected precursor. It is hypothesized is that, like
sca, ed functions to promote N receptor activation in these pre-SOPs. Despite these similarities between sca and ed function, genetic evidence suggests that
they take part in parallel processes. Moreover, Sca and Gp150 are located in late endosomes, whereas Ed is located at the membrane and in early endosomes (Rawlins, 2003).
In vivo and in cultured cells, Ed protein colocalizes very strongly with Dl in cis, both at the membrane and in early endosomes. It is possible that there is a direct
molecular interaction between the two proteins, but no evidence has yet been found for this. Such an association may require Ed-Ed homophilic binding. Nevertheless,
colocalization suggests a close and specific association with Dl-N signalling. One possibility is that Ed promotes Dl function in the 'true' SOP, leading to more efficient
suppression of the emergence of unwanted SOPs. Cis-endocytosis of Dl into the signalling cell is apparently required for activation of the Notch receptor, and one could envisage
that ed may enhance this process in the SOP. This is supported by the colocalization of Ed with N and Dl during N activation as observed in this study's cell culture analysis (Rawlins, 2003).
An alternative is that ed may inhibit Dl activity in recipient (non-SOP) cells. There is evidence that such reduction of Dl activity may promote unidirectional signalling in two ways: (1) it would free an SOP from inhibition by surrounding cells; (2) it has been suggested that Dl in recipient cells antagonizes their response to trans signalling, perhaps by cis association of Dl and N. Therefore, Ed inhibition of this antagonistic function of Dl would make non-SOP cells more vulnerable to signalling from the
SOP. No difference is seen in Dl distribution and level in ed mutant clones, but it is suspected that this might only be apparent in the pre-SOPs. However, after overexpression of Ed, a striking and specific decrease in Dl is observed both at the membrane and in vesicles. Remarkably, this correlates with SOP loss, which is the opposite phenotype to that normally expected for loss of Dl. Thus, Ed function may be connected to the downregulation of Dl in recipient cells. Proteolysis and endocytosis of Dl have both been implicated as causing its downreg