The Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, UK
Author for correspondence (e-mail:
andrew.jarman{at}ed.ac.uk)
Accepted 25 September 2003
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SUMMARY |
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Key words: Notch pathway, Drosophila, Delta, Neurogenesis
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Introduction |
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Much of what is known about Notch signalling stems from studies of sense
organ precursor (SOP) fate specification in Drosophila (reviewed by
Artavanis-Tsakonas and Simpson,
1991; Baker, 2000
).
For the SOPs of the adult external sense (es) organs, the proneural genes
achaete (ac) and scute (sc) are expressed
in the third larval instar imaginal discs in groups of about 20 epidermal
cells - the proneural clusters (PNCs). As development proceeds, proneural
expression is reinforced in three or four PNC cells before being vastly
upregulated in the future SOP and deactivated in the remainder of the cluster
(Cubas et al., 1991
;
Romani et al., 1989
;
Skeath and Carroll, 1991
).
This inhibition results from unidirectional Notch signalling triggered by the
SOP, i.e. lateral inhibition. But before the appearance of an SOP, a state is
thought to exist where signalling is bidirectional between all PNC cells
(mutual inhibition), and this is often equated with prevention of premature
SOP formation or maintenance of competence.
Selection of the SOP requires the progression from bidirectional to
unidirectional signalling - a process that is incompletely understood. One
attractive model is based on the notion that Dl transcription is
repressed by Notch pathway activity. This model suggests that differences in
the initial levels of Dl (and/or N) expression across the PNC results in small
differences in levels of inhibitory signalling. These differences will be
amplified by positive feedback until signal production has been switched off
in all cells except one - the SOP
(Muskavitch, 1994). This is
supported by genetic dose evidence
(Heitzler and Simpson, 1991
).
However, it seems that the levels of N and Dl at the membrane do not
necessarily vary across the PNC (Kooh et
al., 1993
), and Dl transcription is not necessarily regulated by N
activity (Li et al., 2003b
).
Hence, if signalling is initially bidirectional, it is not clear how the SOP
can escape from inhibition from surrounding cells. A number of other
mechanisms have been proposed that `break the symmetry' of signalling within
the PNC and allow a future SOP to emerge. For example, it is proposed that N
protein on the selected precursor becomes resistant to activation by Dl
expressed in the surrounding cells, allowing this cell to inhibit surrounding
cells unidirectionally (Li et al.,
2003b
).
A number of other studies suggest the importance of modulating Dl activity
for promoting differential signalling in the PNC. In particular,
post-translational downregulation of Dl activity on non-SOP cells may allow an
SOP to escape from inhibition and also render the recipient cells more
vulnerable to lateral inhibition (Parks et
al., 2000). Kuzbanian-mediated proteolysis of Dl may fulfil this
function (Mishra-Gorur et al.,
2002
). Dynamin-dependent regulated cis-endocytosis of Dl has also
been strongly implicated (Parks et al.,
2000
), triggered by the ubiquitin ligase Neuralized (Neur)
(Lai et al., 2001
;
Le Borgne and Schweisguth,
2003
).
We investigate the function of the cell adhesion molecule Echinoid (Ed) in
modulating Notch pathway signalling. Ed was originally identified as a
negative regulator of Egfr signalling in eye development
(Bai et al., 2001), where it
restricts R8 precursor specification by preventing inappropriate Egfr-mediated
induction of R8 fate (Rawlins et al.,
2003
; Spencer and Cagan,
2003
). ed mutant flies also bear additional external
sense (es) organs in a pattern reminiscent of mutants of lateral inhibition.
Our investigation reveals that in the context of SOP specification, Ed
modulates Notch pathway signalling, rather than Egfr signalling. We show that
Ed protein associates cytologically with N, and especially Dl, at the membrane
and in endosomes, and that Ed may modulate signalling by influencing the
trafficking/degradation of Dl protein.
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Materials and methods |
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Generation of mitotic clones
Mutant clones were induced using the FLP/FRT method
(Xu and Rubin, 1993).
ed clones were created in a Minute background [y w
FLP122; M(2)Z armLacZ FRT40A/CyO flies obtained from A. Garcia-Bellido]
marked by the absence of ß-galactosidase immunoreactivity and created
using a heat-shock inducible FLP (first instar larvae were
heat-shocked for 1 hour at 37°C). The genotype of the flies was y w
FLP122/y w hsp70-FLP; edlH23 FRT40A/M(2)Z armLacZ FRT40A.
Cell culture
Schneider 2 (S2) cells were maintained at 25°C in Schneider's medium
(Sigma) with 10% fetal bovine serum (Invitrogen). Transient transfections were
performed using Effectene Transfection Reagent (Qiagen) according to the
manufacturer's protocol. Protein expression was induced 24 hours after
transfection with a 35 minute heatshock at 37°C. Cells were harvested
after a further 24 hours. Constructs used were pCaSpeR-hs-N, pCaSpeR-hs-Dl
(provided M. Baron) and pCaSpeR-hs-Ed-Myc, pCaSpeR-hs-Ed-FLAG
(Spencer and Cagan, 2003). All
transient transfection experiments were repeated at least three times with
qualitatively identical results. The numbers in the text are from one
experiment. For aggregation, cells were washed and resuspended in fresh medium
before mixing with gentle rotation in microtiter plates for 4 hours at room
temperature and then processed for immunohistochemistry.
Histology
Scanning electron microscopy (SEM) was performed according to standard
procedures and all scanning electron micrographs were taken at 150x
magnification on a Cambridge Stereoscan 250. Immunohistochemical staining of
third instar larvae was carried out as described
(Rawlins et al., 2003). For
the pupal wing discs, pupae at 24-26 hours after puparium formation (APF) were
dissected in cold PBS and fixed for 1 hour in 4% paraformaldehyde on ice. S2
cells were allowed to adhere to poly-L-Lysine slides for 20 minutes and then
fixed in 3.7% formaldehyde for 3 minutes. Antibody staining followed standard
procedures. Primary antibodies used were guineapig anti-Sens (1:5000; provided
by H. Bellen), guinea-pig anti-Hrs (1:1000; provided by H. Bellen), mouse
anti-Ac (1:10; Developmental Studies Hybridoma Bank [DSHB]), mouse anti-Elav
(1:200; DSHB), mouse anti-Sca (1:200; DSHB), mouse anti-Fasciclin 2 1D4 (1:50;
DSHB), mouse anti-NECD F461.3B (1:50; DSHB), mouse
anti-NICD C17.9C6 (1:50; DSHB), mouse anti-DlECD C594.9B
(1:50; DSHB), rabbit anti-DlECD N2 (1:3000; provided by M.
Muskavitch), guineapig anti-DlICD (1:3000; provided by M.
Muskavitch), mouse anti-E(Spl) 323-2-G (1:2; provided by S. Bray), mouse
anti-Myc (1:300; NEB), rabbit anti-FLAG (1:300; Sigma), mouse
anti-ß-galactosidase (1:250; Promega), rabbit anti-ß-galactosidase
(1:10 000; Cappel), rabbit anti-Ase (1:1000)
(Brand et al., 1993
) and rabbit
anti-Ed (1:5000). For the anti-Ed serum, the ed intracellular domain
reading frame was fused to the GST tag of the pGEX-2T bacterial expression
vector. Using this construct, the protein was expressed, isolated from
bacteria, excised from an SDS-PAGE gel and used to immunise four rabbits.
Serum was preabsorbed and checked for specificity to Ed by western analysis
(1:50 000) and by immunohistochemical staining (1:5000). Secondary antibodies
(1:1000) were obtained from Jackson Laboratories or Molecular Probes. Confocal
images were taken on a Leica TCS SP microscope.
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Results |
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|
|
echinoid interacts genetically with the Notch signalling
pathway but not the Egfr signalling pathway
Culí and others have shown that the EGF receptor plays a role in
signalling between all the cells of the macrochaetae PNCs, which they term
lateral co-operation (Culí et al.,
2001). We have previously demonstrated that R8 twinning in
ed mutants is a consequence of deregulation of Egfr signalling
(Rawlins et al., 2003
;
Spencer and Cagan, 2003
). In
contrast to the R8 phenotype, the ed4.12 bristle
phenotype cannot be consistently modified by reducing Egfr
(EgfrIK35/+) or argos
(aos
7/+) gene dosage
(Table 1). Furthermore, we have
seen no change in the expression of the Egfr pathway targets
pointed-P1 and dpERK in the wing discs of ed mutants
(Gabay et al., 1996
;
Gabay et al., 1997
), even
though such an effect is seen in the eye
(Rawlins et al., 2003
) (data
not shown). These data suggest that the ed bristle phenotype is not
due to a direct effect on lateral cooperation. Consistent with this,
ed mutation affects all thoracic macrochaete locations, whereas only
certain proneural clusters have a strong requirement for Egfr signalling
(Culí et al., 2001
).
In contrast to the above, the ed4.12 bristle
phenotype interacts strongly with mutations in the Notch pathway
(Table 1 and data not shown).
This is most marked for N and Dl themselves. Removing one
copy of N (N55e11) in an
ed4.12 background both increases the number of
SOPs specified and alters their asymmetric divisions to produce four neurons
rather than the wild type es organ (Fig.
2A,B). This is reminiscent of the N null phenotype
(Guo et al., 1996) and occurs
even though null ed mutants alone do not display an asymmetric
division phenotype. Similarly, ed4.12;
Dl/+ flies have additional shaft cells (data not shown). These genetic
interactions suggest that Ed affects the Notch pathway during SOP
specification and asymmetric SOP division.
|
Ed colocalises with N and Dl at the cell surface and in early
endosomes
To address where Ed may function, we raised an antibody to the whole
intracellular domain of Ed. The Ed protein detected by this antibody is
located at the apical cell membrane of all imaginal disc cells and in the
ectoderm of the embryo. It is also present in intracellular vesicles
distributed throughout the cytoplasm in all these cells
(Fig. 3A,F). A transgene with
GFP fused to the C terminus of Ed (UAS-ed-GFP) shows an identical
distribution when expressed in imaginal discs
(Fig. 3B). These vesicles have
not been observed in previous reports (Bai
et al., 2001; Islam et al.,
2003
). However, the antibody used in those experiments was raised
against the extreme N terminus of the protein, which may be cleaved in vivo
and so might not reflect the complete protein distribution
(Bai et al., 2001
;
Spencer and Cagan, 2003
). The
Ed-containing vesicles are not related to Golgi, because only a small fraction
co-label with Fringe-Myc, a Golgi marker
(Munro and Freeman, 2000
)
(Fig. 3C). By contrast, the
majority of Ed vesicles in the apical regions of the cells also express the
early endosomal marker HRS (Lloyd et al.,
2002
) (Fig. 3E).
Furthermore, in hook1 mutants, in which levels of
endocytosis are generally decreased
(Kramer and Phistry, 1996
;
Kramer and Phistry, 1999
),
there was a discernible reduction in the number of Ed-positive vesicles
(Fig. 3F,G). These data suggest
that the majority of the vesicular Ed visible in the cell is in the endocytic
pathway, a large proportion of it being in an early endosomal compartment.
|
Therefore, the colocalisation of Ed with N and Dl in the wing disc is likely to relate to function rather than being coincidental. Despite this, mutation of ed does not affect the frequency of endocytosis of these proteins: the number of NICD, NECD and DlECD vesicles remains unchanged in ed mutant clones (data not shown).
Overexpression of Ed inhibits SOP formation and affects Dl
To determine the effect of overexpression of ed, we expressed
UAS-ed using sca-GAL4 which drives expression in all
proneural clusters. Overexpression of Ed during R8 selection in the eye disc
had no effect (Rawlins et al.,
2003; Spencer and Cagan,
2003
). In notable contrast, sca-GAL4;UAS-ed
flies exhibit many missing bristles (Fig.
1C). Examination of larval wing discs at puparium formation
revealed that very few SOPs have been selected. In the absence of SOPs, Ac
expression persists in many PNCs, owing to lack of lateral inhibition
(Fig. 1J). This phenotype is
qualitatively similar to those seen upon overexpression of E(Spl)C proteins
(Nakao and Campos-Ortega,
1996
). Paradoxically, there is also some bristle duplication when
Ed is overexpressed: occasionally two SOPs can arise from the edge of the same
PNC (Fig. 1C). This may be
explained by heterogeneity of misexpression with sca-Gal4 so that
strong UAS-ed-induced inhibition of neurogenesis in the centre of a
PNC may allow cells at the edge to escape inhibition.
When UAS-ed is expressed in the wing disc using dpp-Gal4, Ed protein is present in almost all of the Dl-positive vesicles, although there is no change in vesicle number. Strikingly, however, the level of Dl immunofluorescence at the cell surface and in the vesicles is markedly reduced compared with that in adjacent wild-type cells (Fig. 4A,B). By contrast, the levels of NECD and NICD are unaffected (Fig. 4C; data not shown). Therefore, inhibition of SOP formation by UAS-ed correlates specifically with loss of Dl immunofluorescence, suggesting that Ed function is more closely related to Dl than N, and that Ed plays a role in the trafficking/degradation of Dl.
|
|
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Seventy-five percent of clone borders do not show twinned SOPs, which we interpret as consistent with the fact that significant lateral inhibition still occurs in null ed alleles. Of these clone borders, 71% show a mutant SOP adjacent to wild-type non-SOP cells and 39% show a wild-type SOP adjacent to mutant non-SOP cells (n=68). The predominance of the former may reflect the greater density of SOPs within the mutant clone.
The behaviour of Echinoid in cell culture in relation to N and
Dl
In vivo, a number of distinct cellular processes are thought to govern N
and Dl signalling and processing (Seto et
al., 2002) and it is not easy to determine whether Ed
colocalisation correlates with a particular event. The S2 cell assay allows a
functional dissection of the various interactions between N and Dl. For
example, S2 cells do not endogenously express N and Dl, but transfection with
N- and Dl-expressing plasmids causes them to aggregate because of their
heterophilic association in trans (Fehon
et al., 1990
). In isolated (non-contacting) S2 cells
co-transfected with both N and Dl, these proteins can be detected at the cell
surface and in vesicles, showing that they can undergo cis-endocytosis
together (Fig. 5B)
(Fehon et al., 1990
). Such
cis-endocytosis of N and Dl is suggested to be linked to inhibition of trans
signal reception in recipient cells (Klueg
et al., 1998
; Kooh et al.,
1993
; Parks et al.,
2000
). When Dl/N expressing cells are also co-transfected with
Ed-FLAG, the latter colocalises with the N/Dl-positive vesicles (74% contain
Ed-FLAG) and the Dl-positive vesicles (66%) but is rarely seen in vesicles on
its own or with N alone (12%) (not shown). This pattern is unchanged upon
association (by aggregation) with an Ed-Myc expressing cell
(Fig. 5C). This is consistent
with the observation in vivo that Ed/NICD/Dl can be present in the
same vesicle (Fig. 3J), and it
implies that this in vivo distribution is most likely to reflect
cis-endocytosis of N, Dl and Ed together from the cell membrane. Consistent
with these results, when Ed-FLAG is co-expressed with Dl alone, the two
proteins colocalise well in vesicles (81% of the Dl-positive vesicles also
contain Ed), whether the cells are solitary
(Fig. 5D) or aggregated (data
not shown). This is true to a lesser extent for N and Ed (23% of the
N-positive vesicles also contain Ed). Therefore in S2 cells, Ed is
cis-endocytosed with Dl, and to a lesser extent N.
Upon association of Dl with N, there is evidence that NECD must
be endocytosed with Dl into the signalling cell in order to trigger
NICD release in recipient cells
(Parks et al., 2000). This
event can be observed in S2 cells: when N-expressing cells are aggregated with
Dl-expressing cells, one can visualise trans-endocytosis of the
NECD and Dl into the signalling cell
(Klueg et al., 1998
;
Parks et al., 2000
). To
investigate whether Ed is associated with Dl during this event, we aggregated
N/Ed-Myc cells with Dl/Ed-FLAG cells and examined vesicle composition for
Ed-FLAG epitope. This showed that more than 95% of the NECD/Dl
vesicles in the signalling (i.e. Dl-expressing) cell also contained Ed-FLAG
(Fig. 5E). In the reciprocal
experiment, N/Ed-FLAG expressing cells were aggregated with Dl/Ed-Myc
expressing cells. In this case, transfer of Ed-FLAG into NECD/Dl
vesicles in the signalling cell was only rarely observed (7%)
(Fig. 5F). In these experiments
full-length N and Dl are occasionally transferred between cells, but Ed is
never associated with them (Fig.
5E,F). These two experiments suggest that, in addition to the
cisendocytosis of Ed with Dl in an isolated cell, Ed is also associated in cis
with the cis-endocytosis of Dl that occurs upon N activation. In summary, in
S2 cells Ed is not associated with all aspects of N/Dl trafficking, rather it
seems to be involved specifically with Dl cis-endocytosis
(Fig. 5G).
In all of the above experiments in S2 cells, the amount of N or Dl endocytosis or N activation was never consistently altered by the presence of Ed and vice versa (not shown). Moreover, in multiple experiments, we found no evidence that colocalisation of Ed and Dl/N represents a direct molecular interaction. Cells transfected with Ed-FLAG could not aggregate with cells transfected with N or Dl. Moreover, association between N-expressing and Dl-expressing cells did not redistribute Ed-FLAG to the cell contact when it was cotransfected in one or other population. Therefore in these assays, Ed does not behave as though binding heterophilically to N or Dl. It is conceivable that such a heterophilic interaction may occur in vivo and that it may be contingent on the homophilic binding of Ed.
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Discussion |
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Echinoid is a modulator of SOP singling out
Ed is not essential for Notch signalling but has a modulatory effect. The
basis of this effect must be relatively subtle, as we find no strongly visible
difference in expression pattern, level, or subcellular localisation of Dl, N
or E(spl) in ed mutant clones. We favour the idea 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 (Li et al., 2003b).
There are two lines of evidence to suggest that Ed functions to inhibit the
transition from PNC cell to SOP. First, no more than four SOPs are selected
from each PNC even in null ed alleles. Second, ed interacts
particularly strongly with ase, which is expressed on the transition
from PNC to SOP. We suggest that the role of ed is analogous to that
proposed for sca (Li et al.,
2003a
). 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 causes all but one of these unwanted SOPs to revert and once
again become responsive to Dl from the selected precursor
(Li et al., 2003a
). Our
hypothesis is that, like sca, ed functions to promote N receptor
activation in these pre-SOPs. Despite these similarities between sca
and ed function, our 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.
Echinoid is closely associated with Delta
In vivo and in cultured cells, Ed protein colocalises 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 we have no
evidence so far for this. Such an association may require Ed-Ed homophilic
binding.
Nevertheless, colocalisation 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.
Cisendocytosis of Dl into the signalling cell is apparently required for
activation of the Notch receptor (Parks et
al., 2000), and one could envisage that ed may enhance
this process in the SOP. This is supported by the colocalisation of Ed with N
and Dl during N activation as observed in our cell culture analysis.
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. First, it would free an SOP
from inhibition by surrounding cells. Second, it has been suggested that Dl in
recipient cells antagonises their response to trans signalling, perhaps by cis
association of Dl and N (Jacobsen et al.,
1998; Sakamoto et al.,
2002
). Therefore, ed inhibition of this antagonistic
function of Dl would make non-SOP cells more vulnerable to signalling from the
SOP. We see no difference in Dl distribution and level in ed mutant
clones, but suspect that this might only be apparent in the pre-SOPs. However,
after overexpression of Ed, we observe a striking and specific decrease in Dl
both at the membrane and in vesicles. Remarkably, this correlates with SOP
loss, which 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 downregulation (Lai et al.,
2001
; Mishra-Gorur et al.,
2002
). It is feasible that Ed promotes one of these processes, for
example by helping to present Dl to Kuzbanian for cleavage.
Ed affects Notch and EGFR pathways independently
ed mutants have twinned R8 photoreceptors in the eye and
additional es organ SOPs everywhere. A priori one would imagine these
phenotypes to have the same genetic and mechanistic basis. They appear,
however, to indicate the interaction of ed interaction with two
different signalling pathways. We, and others, showed that Ed negatively
regulates Egfr signalling (through direct interaction with pathway components)
during R8 specification (Rawlins et al.,
2003; Spencer and Cagan,
2003
). This is in contrast to the role of Ed during es organ
specification, where it modulates Notch pathway signalling. There are several
other reasons for concluding that the R8 and SOP phenotypes of ed
mutants, although superficially similar, have different origins. The latter,
but not the former, is sensitive to overexpression of Ed protein. For R8, this
is explained because Ed is regulated by EGFR post-translationally and so
absolute protein levels are unimportant
(Spencer and Cagan, 2003
).
Sensitivity of SOP singling out to Ed protein levels suggests a different
mechanism is at play. Most strikingly, Ed protein is colocalised extensively
with N and Dl in the wing disc cells, but not in the eye disc, where
interestingly there appears to be very little N and Dl on the cell surfaces
(Kooh et al., 1993
).
Therefore, all this suggests the conclusion that the two phenotypes do indeed
have different origins, and moreover that there are significant differences in
SOP singling out compared with R8 precursor selection.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Present address: Department of Zoology, University of Cambridge, Cambridge
CB2 3EJ, UK
Present address: Division of Biomedical Sciences, George Square, Edinburgh
EH8 9XD, UK
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