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First published online January 13, 2009
doi: 10.1242/10.1242/dev.026054
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
* Author for correspondence (e-mail: mf1{at}mrc-lmb.cam.ac.uk)
Accepted 30 November 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: EGFR, Fasciclin 2, Adhesion, Signalling, Drosophila
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Christofori, 2003), but the
relationship is complex and variable. From first principles, it is obvious
that the degree to which cells stick to each other is likely to influence the
transmission of signals between them. More specifically, there are numerous
examples of specific adhesion complexes at the cell surface that appear to act
as specialised sites of signal transmission (reviewed by
McLachlan and Yap, 2007;
Parsons, 2003). There are also
well-documented examples of specific interactions between growth factor
receptors and adhesion proteins that modulate receptor activity (e.g. reviewed
in Comoglio et al., 2003). In
these latter cases, this may be independent of the adhesion function of the
protein. Beyond the spectrum of different types of relationships, the
interaction between adhesion proteins and signalling can be cooperative or
antagonistic: in some cases adhesion promotes signalling, in others it
inhibits (e.g. Williams et al.,
1994; Francavilla et al.,
2007). Understanding the variety of relationships between
signalling and adhesion is made more difficult by the fact that much of the
information available has relied on cell culture and other in vitro methods.
There are rather few cases where the in vivo significance of interactions
between signalling and adhesion proteins is clear.
We have used Drosophila genetics to search for physiologically
significant regulators of the epidermal growth factor receptor (EGFR)
signalling pathway. The Drosophila EGFR is the orthologue of the four
ErbB receptors in mammals, and has multiple functions in development
(Shilo, 2003;
Domínguez et al.,
1998). In different contexts, EGFR signalling can trigger cellular
responses as varied as differentiation, division, survival and migration. A
consequence of this pleiotropy is that regulation of signalling strength and
location must be precise. Moreover, signalling pathways appear to incorporate
mechanisms of robustness against environmental perturbation. Our long-term
goal is to understand the molecular machinery that provides these stringent
control properties. In addition to revealing the logic of normal development,
deregulation of ErbB activity is implicated in many human diseases, especially
cancer (Uberall et al., 2008),
further emphasising the importance of understanding the control of this
pathway. An advantage of the forward genetic approach that we have used is
that it makes no assumptions about the types of proteins needed for
regulation; instead, it relies on random mutagenesis to reveal significant
players - however novel or unexpected. It therefore complements more focused
biochemical approaches. We have used genetic modifier screens in the
Drosophila eye as a tool to identify novel regulators of EGFR
signalling (Casci et al., 1999;
Charroux et al., 2006). The eye
develops in a well-characterised and stereotypical way
(Wolff and Ready, 1993), and,
because it is dispensable for viability, at least in the laboratory, it
provides an excellent platform for genetic screening.
Many intracellular transducers and regulators that act downstream of the
EGFR have been identified, but much less is known about the control of earlier
events of signalling: the generation, transmission and reception of the
ligand. Here, we report the identification of Fasciclin 2 in a genetic screen
that was designed to focus on these earlier stages. The Fasciclin 2 (Fas2)
protein is the Drosophila orthologue of the mammalian neural cell
adhesion molecule (NCAM) family
(Grenningloh et al., 1991;
Cunningham et al., 1987), and
our results show that it acts specifically to inhibit EGFR signalling during
the normal development of the Drosophila eye, notum and wing.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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7,
rhomboid-15, fas2eb112 sn
FRT19A/FM7c (Grenningloh et al.,
1991) (gift from C. Fabre); fas2e76
(Grenningloh et al., 1991)
(gift from L. Garcia Alonso); fas2G0293/FM7c,
fas2G0336/FM7c (from Bloomington Drosophila Stock Centre);
w ubiGFP M(1)ospFRT19A; A59/TM6b (this study); XA12
(R7-lacZ), P(w+, X81) rho-lacZ, spiA14FRT40A/Cyo,
Dlrev10, GMRSu(H)DN, HairlessP141 (gifts from S.
Bray); F76e, sev-wg, UAS-cadi,en-Gal4 (gifts from M. Bienz);
UAS-Smo5A,C765-Gal4 (a gift from S. Cohen); and
GMR>
-btl, dof, trk5, dlgG0276,
dlgG0342, dlgG0456, Nrgl7, Nrgl4,
NrgG0099, NrgG0413, lgl4,
scribj7B3.
Mitotic clones in the eye and wing discs were induced by the FLP/FRT
technique (Xu and Rubin, 1993)
in Minute and non-Minute background
(Morata and Ripoll, 1975).
Recombination was induced 48-72 hours after egg laying by a 60 minute heat
shock at 37°C or by eyeless-induced FLP activity. Mutant clones
were marked as appropriate by the absence of GFP or β-galactosidase
(β-gal) antibody staining. The following genotypes of larvae were used
for generating mutant clones: fas2eb112 sn FRT19A/arm-lacZ
FRT19A; eyflp/+ and fas2eb112 sn FRT19A/ubi-GFP
M(1)ospFRT19A; MKRShsflp/+ (Minute background)
Immunostaining
Imaginal discs and pupal retinas (aged 40 hours at 25°C after pupation)
were stained as described previously (Gaul
et al., 1992). Primary antibodies used were: mouse anti-Fas2 1D4
(1:50), rat anti-Elav (1:200), mouse anti-Cut (1:100), mouse anti-Prospero
(1:50), mouse anti-Yan (1:100), mouse anti-Achaete (1:10) (all from
Developmental Studies Hybridoma Bank), rabbit anti-Armadillo (1:100) (a gift
from M. de la Roche), guinea pig anti-Senseless (1:1000) (gift from H.
Bellen), rabbit anti-pMad (1:200) (gift from E. Laufer and C. Heldin), rabbit
anti-β-galactosidase (1:1000) (Cappel), rabbit anti-GFP (1:200) (Sigma).
The appropriate fluorescently conjugated secondary antibodies from Molecular
Probes and Jackson ImmunoResearch were used.
Confocal imaging and three-dimensional reconstruction
For three-dimensional reconstruction, discs were mounted under a coverslip
supported by two strips of double-sided adhesive tape using Fluoromount-G
(Southern Biotech). Discs were analysed with a BioRad Radiance 2100 laser
scanning confocal microscope. Z-series were projected for three dimensional
reconstruction using Volocity 2.5.1 software. All images were analysed using
Adobe Photoshop.
Fluorescence intensity quantification
Fluorescence (pixel) intensities of confocal images were quantified using
MetaMorph.
In situ hybridisation
The first 1.5 kb of the fas2-coding sequence was amplified from
FasIIPA cDNA (a kind gift from Michael Hortsch) (primers were F,
5'-ATGGGTGAATTGCCGCCAAATTC-3' and R,
5'-AGAGTAATACTGCCTCGTTACGGG-3') and cloned into pCRBlunt IITOPO
vector using standard techniques. DIG-labelled RNA antisense and sense probes
were transcribed using Sp6 and T7 promoters, respectively, using
manufacturer's instructions from Roche. In situ hybridisation on imaginal
discs were carried out using standard procedures
(Cubas et al., 1991).
Embryonic cuticle preparations
Overnight embryo collections were allowed to age for 24 hours. Embryos were
dechorionated in 50% bleach, removed from their vitelline membranes, and
mounted in 1:1 Hoyer's mountant:lactic acid. Cuticles were viewed under
dark-field optics.
Scanning electron microscopy of adult eyes
Flies were frozen for at least 1 hour at -80°C, mounted onto aluminium
electron microscope specimen stubs, and coated with 20 nm of a gold-palladium
mixture. Samples were viewed on a Philips XL30 scanning electron
microscope.
Photographs of adult flies
Flies were positioned appropriately and examined under a dissecting
microscope. Images were taken at roughly 35 different focal planes with a
Nikon D3 digital camera. The planes were then stacked using Helicon focus
software.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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); and downregulated by the overexpression of Argos, an
inhibitor that binds soluble ligand (Klein
et al., 2004; Klein et al.,
2008), preventing it from activating the receptor. The logic of
using these two conditions is based on our experience that modifier screens
are most efficient at identifying pathway components close to the site of
perturbation. Rhomboid 1 and Argos act in ligand production and transmission,
therefore we hoped to identify other components at this level of the pathway.
For rigour, candidates were selected only if they interacted with both
conditions and in opposite directions: that is, a suppressor of one needed to
enhance the other. In one iteration of this strategy, a collection of
approximately 350 P-elements on the X-chromosome was screened. Of these, 50
interacted with either GMR-argos or sevrhomboid-1, of which
20 were identified as interacting with both in opposite directions. These 20
candidates were then narrowed down as follows: 10 were revertable by precise
excision of the P-element (confirming that the P-element identifies the
relevant gene), of which three showed consistent interactions with other
components of the EGFR pathway; only one of these three also had a
loss-of-function phenotype consistent with a role in EGFR signalling. This
single gene, selected by this hierarchical triage, was an allele of the
fasciclin 2 (fas2G0293) gene. It was identified
as a suppressor of GMR-argos and as an enhancer of
sev-rhomboid-1 (Fig.
1A-D). These interactions were confirmed with other alleles of
fas2, including fas2eb112
(Fig. 1E), an independently
derived null allele (Grenningloh et al.,
1991), and suggested that Fas2 might inhibit EGFR signalling. Fas2
is a member of the immunoglobulin superfamily and is the Drosophila
orthologue of the conserved NCAM family of neural cell-adhesion molecules
(Grenningloh et al., 1991). To
investigate further the possibility that Fas2 inhibits EGFR signalling in the
Drosophila eye, we examined genetic interactions with other
components of the pathway. Consistent with the screen results, halving the
dose of fas2 enhanced the rough eye caused by overexpression of
Egfr itself (Fig.
1F,G) and suppressed the rough eye caused by the overexpression of
sprouty, another inhibitor of the pathway
(Fig. 1H,I).
The fas2 gene is expressed in a subset of neuronal cells in
embryonic and post-embryonic nervous system. We examined the distribution of
transcripts in the third instar imaginal eye disc by in situ hybridisation
(Fig. 2A). fas2 was
not expressed in the undifferentiated cells anterior to the morphogenetic
furrow but appeared strongly at the furrow; it then decreased for a few rows
of ommatidia before strengthening again towards the posterior of the disc.
Antibody staining confirmed this dynamic developmental pattern for the Fas2
protein (Fig. 2B): it was
strongly upregulated just posterior to the furrow, prior to the expression of
Senseless, the earliest marker of the R8 photoreceptor that seeds each
ommatidium (Fig. 2C). After the
initial strong expression in the preclusters, Fas2 remained expressed at a low
level in distinct ommatidial cluster patterns in the cell membranes of most
photoreceptor cells. Eventually, one photoreceptor accumulated elevated levels
of Fas2, and co-staining with an R7-expressing β-gal reporter line
XA12 (Hart et al.,
1990), showed this to be the R7 photoreceptor (arrows,
Fig. 2G). Significantly,
although the bulk of Fas2 protein was detected basolaterally
(Fig. 2D-F), in those cells
with elevated levels (i.e. just posterior to the furrow and, later, R7s), we
observed Fas2 staining in the apical compartment, where EGFR signalling occurs
(arrows Fig. 2D-F).
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). We examined the pupal retinas from these rough eyes
(Fig. 3B) and found that
photoreceptor number was disrupted: 5% of ommatidia (n=158) had at
least one extra photoreceptor [0% in wild type (WT),
Fig. 3A]; 4% had fewer
photoreceptors (0% in wild type); and about 4% of clusters appeared by
location and photoreceptor number to be satellite `mini-clusters' (0% in wild
type, see below for discussion of these, and
Fig. 3F' for an example).
Cone cell numbers were also disrupted: 3% of ommatidia (n=158) had
extra cone cells (0% in wild type); 3% had fewer cone cells (0% in wild type);
and 4% contained fewer cone cells in `mini-clusters' (0% in wild type). To determine whether the rough eye phenotype of these fas2eb112/fas2e76 transheterozygotes could be modified by changing levels of EGFR signalling, we examined the phenotype of additionally halving the dose of argos, an EGFR-specific inhibitor. At 18°C, there were very few survivors, so the test was carried out at 20°C where viability was improved slightly. The fas2eb112/fas2e76 phenotype was only very mildly rough at this temperature (Fig. 3C), but this was significantly enhanced by halving the dose of argos (Fig. 3E). This is consistent with the interactions described above, and suggests that reduction of Fas2 leads to upregulated EGFR signalling.
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;
Casci et al., 1999); moreover,
hyperactive EGFR activity has been shown directly to trigger the
differentiation of photoreceptors in the absence of a founding R8 cell
(Domínguez et al.,
1998). The presence of these ectopic photoreceptor clusters is
therefore a hallmark of EGFR hyperactivity. In further correspondence with the
pupal phenotype, ommatidia with extra photoreceptors were also observed,
another phenotype of excess EGFR signalling. By contrast, unlike in pupal
retinas, we were unable to observe ommatidia at this stage with too few
photoreceptors, suggesting that the observed loss of photoreceptors may occur
later in development.
We used cell-type specific markers to investigate the identity of the extra
photoreceptors. In argos mutants, there is a frequent transformation
of non-neuronal `mystery cells' into extra R3/4 type photoreceptors, some of
which break away to form ectopic mini-clusters; more rarely extra R7 cells are
recruited to existing ommatidia (Freeman
et al., 1992). Conversely, the extra cells in sprouty
mutants are predominantly R7 type, although extra outer photoreceptors,
forming ectopic mini-clusters, also occur
(Casci et al., 1999).
Photoreceptors in the ectopic mini-clusters in fas2 mutants did not
express markers that identified them as having a specific photoreceptor
subtype, but the location of their initiation close behind the furrow and
their positioning with respect to the ommatidia
(Fig. 4A), is very similar to
the ectopic clusters seen in argos and sprouty mutants. We
therefore infer that they most closely resemble outer photoreceptors. In those
fas2- ommatidia that show excess recruitment, the
additional photoreceptors (Elav positive) were stained by anti-Prospero [an R7
and cone cell marker (Kauffmann et al.,
1996)] and were therefore R7-like
(Fig. 4C-D').
Interestingly, genotypically wild-type ectopic R7 cells were found at the
borders of clones (Fig.
4D-D'); that is, a wild-type cell could adopt an abnormal
fate if adjacent to fas2 mutant cells, an indication of a
non-autonomous effect. In summary, we detect two types of extra photoreceptors
in fas2- clones: those that resemble outer photoreceptors,
forming the mini-clusters; and R7-like cells, recruited into otherwise normal
ommatidia.
The recruitment of ectopic clusters and excess photoreceptors associated
with loss of fas2 are specific characteristics of EGFR hyperactivity
and therefore strongly support the case that Fas2 acts to inhibit EGFR
signalling in the eye. An even more direct readout is the stability of the Yan
protein, a transcriptional repressor of EGFR targets that is targeted for
degradation by EGFR signalling (O'Neill et
al., 1994; Rebay and Rubin,
1995). fas2-null clones showed a clear and reproducible,
albeit modest, reduction in Yan protein levels, implying abnormally high
levels of EGFR activity (Fig.
4E-G). Consistent with the Fas2 expression pattern, this phenotype
was apparent only close to the furrow and towards the posterior of the eye
disc: no effect was seen in the intervening zone of low Fas2 expression. The
most prominent manifestation of this phenotype was the enlarged `holes' in the
Yan expression pattern, where Yan was clearly degraded more extensively than
in neighbouring wild-type tissue (Fig.
4E'-G). This pattern of excess degradation presumably
relates to the normal high level of Fas2 expression being limited, at this
stage in eye development, to the R7 cells. Therefore, loss of Fas2 would be
expected primarily to affect R7 precursors and their neighbours (recall the
local non-autonomous effects we observed in fas2- clones).
To measure the reduction in Yan staining, we quantified the average
fluorescent intensities of GFP and Yan staining across an area of wild-type
and mutant tissue at the same developmental stages of nine different
fas2- clones (example marked in
Fig. 4H). This revealed a
significant reduction in both overall Yan levels and the troughs of staining
corresponding to the `holes' in the staining pattern
(Fig. 4I).
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Fasciclin 2 inhibits EGFR signalling in the developing notum and wing
The wing imaginal disc gives rise to the adult wing and the notum, and in
both these structures the EGFR has well defined roles
(Guichard et al., 1999;
Culi et al., 2001). We
therefore analysed Fas2 expression in the developing wing disc, both by RNA in
situ hybridisation (Fig. 5A)
and by antibody expression. Fas2 is expressed in the proneural clusters in the
notum region of the wing disc (Fig.
5B), as well as in the wing vein and wing margin primordia
(Fig. 5C); we also detected
expression in proximal regions of the wing blade and the hinge region
(Fig. 5B,C). The overlap in
some cells between the expression of Fas2 and Rhomboid-1 is consistent with a
functional relationship between EGFR signalling and Fas2. It should be
emphasised, however, that the Fas2 expression is much wider than the domain of
Rhomboid-1 expression, implying that other signals also participate in the
determination of the Fas2 domain.
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)
(Fig. 5F). Furthermore,
expression of a constitutively active form of the EGFR produces expanded
proneural clusters (Y.M. and M.F., unpublished). Consistent with the idea that
Fas2 is an inhibitor of EGFR signalling, null fas2 clones had
expanded proneural clusters and ectopic SOPs
(Fig. 5G-J'). We note
that it has previously been reported that Fas2 is required for the formation
of specific proneural clusters in the developing head
(Garcia-Alonso et al., 1995);
this apparent opposite function is presumably an example of the context
dependence of signalling. Another characteristic phenotype of EGFR
hyperactivity is the formation of extra wing vein cells
(Sturtevant et al., 1993);
again, these are observed in fas2 clones (see Fig. S1 in the
supplementary material). Overall, these results in the notum and wing all
support the conclusion that Fas2 acts to inhibit EGFR signalling in normal
development, and they imply that it is not an eye-specific function. We also noted that the wings of flies with the viable fas2 hypomorphic genotype appeared abnormally elongated when grown at 18°C. By measuring the ratio of their length to breadth, it was confirmed that fas2- wings had a length to breadth ratio of 2.24±0.05 (mean±s.d.; n=19), significantly different (P<0.0001) from 2.13±0.03 in wild-type wings (also grown at 18°C, n=19; see Fig. S2 in the supplementary material). Although EGFR activity is implicated in the control of cell proliferation in imaginal discs, it has not previously been reported to control wing shape. We examined this directly and found that overexpression of wild-type EGFR, a condition that only moderately increases signalling, also leads to significant (P<0.0001) wing elongation, albeit less pronounced (2.18±0.02, n=20). Higher levels of EGFR signalling caused widespread transformation of intervein cells to ectopic veins (not shown), making the wings too abnormal to determine change in shape.
Feedback regulation by Fas2
Regulatory precision of the Drosophila EGFR pathway depends on
multiple inhibitors. The expression of at least three of these, Argos, Sprouty
and Kekkon-1, depends on EGFR signalling, meaning that they participate in a
negative-feedback control strategy that limits the extent and/or strength of
signalling (Shilo, 2003).
Here, we report that Fas2 is a new inhibitor of EGFR signalling: is it also
part of a negative-feedback loop? Overexpression of the EGFR activator
Rhomboid-1 in the eye or the wing, under the respective control of the
sevenless enhancer or Dpp-Gal4, did not induce detectable
upregulation of Fas2, implying that EGFR signalling is not a rate-limiting
determinant of Fas2 expression in these cells. However, loss of EGFR activity,
in spitz mutant clones, caused a failure of Fas2 upregulation in the
developing eye disc (31/31 clones) (Fig.
6), demonstrating a formal dependence on EGFR activity, and
consequently the existence of a potential feedback loop. This conclusion needs
to be qualified, however, because, in the absence of EGFR signalling, disc
cells are not recruited to become photoreceptors, so the dependence may be
indirect: the cells within the clones do not become photoreceptors.
Nevertheless, the dependence on EGFR activity implies that Fas2 expression may
participate in a self-limiting mechanism for EGFR signalling.
Specificity of interaction between EGFR and Fas2
Fas2 is a homophilic adhesion molecule, and disrupting the degree of
contact between cells may have quite broad and non-specific effects. We
therefore tested whether mutations in fas2 were also able to disrupt
signalling by other major pathways. To analyse the Notch, Wingless and
Hedgehog pathways, we used several genetically sensitised conditions,
analogous to our original screen for modifiers of EGFR signalling, to ask
whether halving the dose of fas2 modulated pathway activity. In no
case was any phenotypic modification observed (see Fig. S3 in the
supplementary material). We also used a similar logic to test whether
signalling by two other receptor tyrosine kinases, Torso and FGFR, was
affected by reduction of Fas2. No modification of Torso activity was observed
(see Fig. S3 in the supplementary material). By contrast, halving the dose of
fas2 suppressed the phenotype caused by misexpression in the eye of
an activated form of the FGF receptor Breathless in conjunction with
Downstream-of-FGF-receptor (Dof) (Zhu et
al., 2005) (see Fig. S3 in the supplementary material). Note,
however, that this genetic interaction is the opposite of the effect we see
with the EGFR: it implies that Fas2 is a positive regulator of the FGFR
pathway, which is consistent with other contexts where it has been reported
that Fas2 induces FGFR activity to stimulate neurite outgrowth
(Forni et al., 2004). In the
absence of a suitable genetically sensitised condition for assaying Dpp
modulation, we assayed the levels of phosphorylation of the Dpp transducer Mad
in fas2-null clones in the wing disc. We were unable to see any
changes (see Fig. S3 in the supplementary material). These experiments do not
rule out subtle effects of Fas2 on signalling by these pathways, but they do
imply that a robust and consistent inhibitory effect is specific to EGFR
signalling.
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),
were also tested in the screen. Halving the dose of Discs-large, Neuroglian,
Lethal-giant-larvae and Scribble did not modify either of the interaction
conditions (overexpressed EGFR or argos, data not shown),
implying that the effect of Fas2 on EGFR signalling is distinct from its
adhesion function in basolateral junctions. We also found that Capricious and
Tartan (Milan et al., 2001),
two LRR repeat proteins related to Kekkon-1, a known inhibitor of the EGFR
(Ghiglione et al., 1999),
failed to modulate EGFR signalling (Mao et
al., 2008). On the basis of these results, we conclude that the
relationship between Fas2 and the EGFR is specific: Fas2 does not
substantially inhibit a number of other common signalling pathways, including
those controlled by other receptor tyrosine kinases; and several other
adhesion components do not substantially modulate EGFR signalling. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Casci et al.,
1999; Ghiglione et al.,
1999; Bai et al.,
2001; Gaul et al.,
1992). The explanation for the variation in strength and detail of
phenotype is that each of the inhibitors has a different molecular mechanism
and site of action in the pathway, as well as different sites of expression.
For example, Argos is specific to the EGFR and is a diffusible molecule that
sequesters ligand. By contrast, Sprouty, a cytoplasmic protein, inhibits a
range of receptor tyrosine kinases, whereas Echinoid and Kekkon-1 are cell
surface proteins that bind directly to the EGFR. It is evident that EGFR
regulation depends on a patchwork of overlapping effects of multiple different
types of modulators, each of which has greater or less importance in different
developmental contexts. Presumably, this network of regulators underlies the
observed precision and robustness of signalling.
Loss of Fas2 in the eye triggers at least two distinct types of extra
photoreceptor recruitment. The ectopic mini-clusters appear at the same time
that the normal outer photoreceptors are recruited and, by analogy with
argos mutations (Freeman et al.,
1992), we believe them to be caused by transformation of the
`mystery cells'. In normal development these form part of the precluster, but
are ejected prior to the onset of photoreceptor differentiation. It is also
possible that some of the mini-clusters are derived from de novo photoreceptor
determination occurring in undifferentiated interommatidial cells, which is
known to be triggered by excess EGFR activity
(Domínguez et al.,
1998). The second recognisable type of extra photoreceptors are
the R7-like, Prospero-positive cells. These are presumably the product of
abnormal recruitment of cone cell precursors as R7s, a switch of fates within
the R7 equivalence group, which is sensitive to altered levels of receptor
tyrosine kinase signalling (Tomlinson and
Ready, 1986; Van Vactor et
al., 1991; Freeman,
1996).
Our genetic data do not reveal a molecular mechanism for the inhibition of EGFR by Fas2 - that will require future biochemical analysis - but its location at the plasma membrane and the non-autonomy we detected at the border of mutant clones point to three classes of model:
homologues
Spitz or Keren, for example by direct sequestration of the mature ligand.
Precedence leads us to favour the second model. Two other adhesion
proteins, Kekkon-1 (Ghiglione et al.,
1999) and Echinoid (Spencer
and Cagan, 2003), interact directly with the EGFR. Similarly,
mammalian E-cadherin can inhibit the EGFR by direct binding
(Qian et al., 2004). Of
particular relevance to this work, it has recently been reported that
mammalian EGFR can be inhibited by NCAM, the Fas2 orthologue
(Povlsen et al., 2008). In
these experiments using explanted mouse neurons combined with transfected
mammalian cell lines, NCAM stimulates neurite outgrowth by blocking EGFR
function. Preliminary results lead the authors to favour a mechanism of
NCAM-induced downregulation of EGFR levels, although direct parallels with our
work are difficult to draw because the cytoplasmic domains of NCAM and Fas2
are not similar.
Beyond the evidence for inhibition of the EGFR described here and in the
recent paper discussed above, Fas2/NCAM has now been implicated in several
other signalling systems. The best characterised of these is an interaction
with FGFR signalling, where, both in Drosophila and mammals, FGFR
activity is required for Fas2/NCAM induced neurite outgrowth
(Williams et al., 1994;
Forni et al., 2004) and direct
binding of NCAM activates FGFR (Kiselyov
et al., 2003; Christensen et
al., 2006). By contrast, and an illustration of the context
dependence of such interactions, it has also recently been reported that NCAM
can inhibit FGFR activation by its ligand FGF
(Francavilla et al., 2007).
Less well studied links between NCAM and growth factors include the
observation that NCAM can act as a signalling receptor for GDNF
(Paratcha et al., 2003), and
that it participates in the response of oligodendrocyte precursors to PDGF
(Zhang et al., 2004). The work
we report here is the first genetic evidence to imply a role for Fas2 in the
physiological inhibition of EGFR activity. It is important to set this
discussion in the context of the well established role of Fas2/NCAM as a
neural cell-adhesion molecule, with roles in axonal growth and pathfinding, as
well as in synaptic maturation
(Grenningloh et al., 1991);
Schuster et al., 1996).
Overall, it is becoming clear that the EGFR pathway is regulated by multiple partially overlapping mechanisms, presumably because of the importance of regulatory precision and robustness of such a central and pleiotropic pathway. Notably, negative-feedback control is a recurring theme. Much less is known about physiologically significant regulators of EGFR signalling in mammals, and it will be interesting to determine whether feedback control is a conserved strategy. As there are many other signalling pathways and adhesion proteins that contribute to normal development, the total potential number of regulatory interactions between these key cell surface proteins is enormous and, indeed, many have been observed in vivo and in vitro. Of course, some of these might not occur in normal biological contexts, emphasising the value of a genetic approach to revealing which relationships between adhesion proteins and signalling pathways are physiologically relevant.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/3/473/DC1
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Bai, J., Chiu, W., Wang, J., Tzeng, T., Perrimon, N. and Hsu,
J. (2001). The cell adhesion molecule Echinoid defines a new
pathway that antagonizes the Drosophila EGF receptor signaling pathway.
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Casci, T., Vinós, J. and Freeman, M.
(1999). Sprouty, an intracellular inhibitor of Ras signalling.
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