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 14 May 2003
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SUMMARY |
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Key words: EGF receptor, Drosophila, Photoreceptor, R8
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INTRODUCTION |
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The patterning of the Drosophila compound eye as a hexagonal array
of ommatidia depends on precise spacing of the ommatidia, which in turn relies
on selection and patterning of founding R8 photoreceptor cells in a regular
grid within the undifferentiated retinal ectoderm. This requires complex cell
interactions that are incompletely understood, but involve an interplay
between cell signalling and the proneural gene atonal (ato).
ato encodes a bHLH transcription factor that endows cells with R8
competence. R8 patterning is a progressive process and this is reflected in
the evolution of the Ato expression pattern. In the eye imaginal disc, Ato is
initially expressed in a stripe of cells just anterior to the morphogenetic
furrow as it traverses the unpatterned ectoderm
(Jarman et al., 1994). As the
wave of expression moves on, the stripe becomes broken into evenly spaced
clusters of cells, with Ato expression inhibited between them. Each of these
`intermediate groups' (IGs) is analogous to the proneural clusters of bristle
SOPs (Jarman et al., 1995
).
Within each IG, Ato expression is then resolved to a solitary R8 precursor
before being completely downregulated
(Dokucu et al., 1996
).
Complex cellular interactions regulate ato during IG patterning
and R8 selection. The regular interruption of ato expression that
gives rise to a nascent row of IGs depends on inhibitory signalling from the
previous row of spaced IGs. Mutations in genes involved in this process result
in irregular and denser IG spacing, as is seen for the secreted molecule
Scabrous (Sca) (Ellis et al.,
1994). A number of studies have implicated Egfr/Raf/Ras signalling
in IG spacing, possibly in cooperation with Notch
(Dominguez et al., 1998
;
Spencer et al., 1998
;
Chen and Chien, 1999
;
Baonza et al., 2001
), although
this conclusion is not universally accepted
(Kumar et al., 1998
).
R8 selection within the IGs is a separate event from IG patterning,
although the two are often confused because some genes are required in both
processes, including Notch and sca
(Baker et al., 1996). In
principle, R8 selection is akin to sense organ precursor (SOP) formation (e.g.
for sensory bristles) in that it involves Notch-mediated lateral inhibition
within groups of competent cells defined by ato expression (the IGs
being equivalent to SOP proneural clusters). Nevertheless, there is evidence
for at least two discrete steps in the refinement process that reveals
unexpected (and unaccounted for) complexity. The first step is the refinement
of Ato expression and R8 competence to a group of three cells distinguished
initially by virtue of their raised nuclei
(Dokucu et al., 1996
) and then
by low level expression of the R8 marker encoded by senseless
(sens) (Frankfort et al.,
2001
). Dokucu and colleagues named this the R8 equivalence group
(Dokucu et al., 1996
). The
second step is the restriction of R8 fate to one of these three cells,
coinciding with restriction of ato and sens expression. The
equivalence group represents a group of cells that are uniquely primed to take
on an R8 fate. This is apparent in a number of gene mutations that result in
extra R8 cells specifically from the equivalence group rather than the IG as a
whole, as observed in sca (Ellis
et al., 1994
) and rough (ro) mutants, and also
after experimental overexpression of ato
(Dokucu et al., 1996
;
White and Jarman, 2000
). In
these mutations, the normally isolated R8 cells are frequently replaced by
twins and triplets the so-called `R8 twinning' phenotype.
The role of Egfr signalling in R8 selection has been contentious because of
contradictory evidence. Most studies have concluded that while Egfr/Raf/Ras
signalling may be required for correct IG spacing, such signalling is not
absolutely required for a cell to take on an R8 fate
(Dominguez et al., 1998;
Kumar et al., 1998
). For
example, R8 selection can occur within Egfr mutant clones, albeit aberrantly
(Dominguez et al., 1998
).
Nevertheless, Egfr signalling appears to be active during R8 selection
(Kumar et al., 1998
;
Wasserman et al., 2000
) and
other evidence has been presented that suggests Egfr/Raf/Ras signalling is
required for R8 fate (Spencer et al.,
1998
). Thus, R8 selection within the equivalence group is poorly
understood.
We recently described the effect of overexpressing ato in the
developing R8 precursors using an R8 specific Gal4 driver
(109-68Gal4) to drive UAS-ato
(ato109-68) (White and
Jarman, 2000). Although such overexpression does not alter the
expression pattern of ato beyond boosting and extending it within R8
cells, ato109-68 exhibited several defects in eye
development. One of these defects was R8 twinning, indicating failure of R8
resolution within the equivalence group. This is unexpected because
overexpressing ato in R8 should increase Notch-mediated lateral
inhibition, not reduce it. This non-autonomous effect therefore suggests that
undefined signalling mechanisms that impinge on R8 resolution are being
affected by ato misexpression.
To investigate the process of R8 selection further, we used
ato109-68 as the basis of a screen for genetic modifiers
to isolate mutations that affect R8 resolution. We isolated a mutation of
echinoid (ed), recently described as encoding an L1-like
cell adhesion molecule (Bai et al.,
2001). The ed mutation dominantly enhances the R8
twinning defect of ato109-68 and, contrary to a previous
report, also exhibits severe R8 twinning as a homozygote. Unexpectedly, our
investigation of this phenotype revealed strong indications that the R8
twinning results from derepression of Egfr signalling within the R8
equivalence group causing inappropriate inductive interactions between these
cells. We suggest that ed acts as a novel Egfr antagonist in this
context to downregulate Egfr signalling, and thereby modulate the outcome of
signalling.
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MATERIALS AND METHODS |
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Mutagenesis
To obtain genetic modifiers of ato109-68, male
OrR flies were fed 25 or 30 mM EMS and then mated to
ato109-68/CyO females. The eyes of the F1 progeny were
examined under a dissecting microscope for enhancement or suppression of the
rough eye phenotype. Potential modifiers were backcrossed to
ato109-68/CyO and the eyes of the F2 progeny rescored.
Further crosses were then performed to determine genetic linkage and establish
a balanced stock. Similarly, to obtain further ed alleles, male
OrR flies were fed 25 or 30 mM EMS and then mated to
ed4.12/CyO females.
Generation of mitotic clones
Mutant clones were induced using the FLP/FRT method
(Xu and Rubin, 1993).
ed and spi clones were marked by the absence of nlsGFP
(2xnlsGFP, FRT40A flies obtained from A. Gonzalez-Reyes) and induced
by eyelessFLP (Newsome et al.,
2000
). Flies had the following genotypes: y w eyFLP;
edlH23 FRT40A/2xnlsGFP FRT40A, y w eyFLP; ed4.4
FRT40A/2xnlsGFP FRT40A, y w eyFLP; ed6.1 FRT40A/2xnlsGFP FRT40A, y
w eyFLP; spiSC2 FRT40A/2xnlsGFP FRT40A or y w eyFLP;
ed4.12 spiSC2 FRT40A/ed4.12 2xnlsGFP FRT40A.
Egfr clones were induced in a Minute background, marked by the
absence of ß-galactosidase immunoreactivity and created using a heatshock
inducible FLP (first instar larvae were heat-shocked for 1 hour at
37°C). Egfr clones were induced in flies of the following
genotypes: y w hsp70-FLP; FRT42D EgfrIK35/FRT42D arm-lacZ
M(2)53 (Dominguez et al.,
1998
), y w hsp70-FLP; ed4.12 FRT42D
EgfrIK35/ed4.12 FRT42D arm-lacZ M(2)53 or y w
hsp70-FLP; FRT42D EgfrIK35 scaBP2/FRT42D
arm-lacZ M(2)53.
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. For immunohistochemistry
staining, eye-antennal imaginal discs were dissected from wandering third
instar larvae and fixed in 3.7% formaldehyde (10-15 minutes). Incubations with
primary and secondary antibodies were performed according to standard
procedures. Primary antibodies used were affinity purified rabbit anti-Ato
(1:2000), mouse anti-Boss (1:200; provided by S. L. Zipursky), guinea-pig
anti-Sens (1:5000; provided by H. Bellen), mouse anti-Sca [1:200;
Developmental Biology Hybridoma Bank (DBHB), Iowa, USA], mouse anti-Ro (1:200;
DBHB), mouse anti E(spl) 323-2-G (1:2; provided by Sarah Bray), rabbit
anti-ß-galactosidase (1:10000; Cappel) and mouse anti-dpErk (1:500;
Sigma). Secondary antibodies (1:1000) were obtained from Jackson Laboratories
or Molecular Probes. Confocal fluorescence images were taken on a Leica TCS SP
microscope.
For mRNA in situ hybridisation eye-antennal imaginal discs were dissected from wandering third instar larvae, fixed in 3.7% formaldehyde (1 hour) and then dehydrated in ethanol and stored at -20°C until use. DIG-labelled mRNA probes were in vitro transcribed using a DIG RNA labelling kit (Roche). The DIG label was detected using a sheep anti-DIG alkaline phosphatase coupled antibody. Light microscope images were taken on an Olympus AX70 microscope.
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RESULTS |
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ed is distinct from other mutations that cause R8
twinning
Mutation of sca or ro also results in an R8 twinning
phenotype. ro is a negative regulator of ato that is
expressed in cells that do not take on the R8 fate
(Dokucu et al., 1996). Sca
protein is normally secreted by the cells of the IG at a low level, and then
from the selected R8 cell at a high level, probably preventing R8 twinning by
interacting with the Notch receptor during lateral inhibition
(Baker et al., 1990
;
Mlodzik et al., 1990
;
Powell et al., 2001
). R8
twinning is not completely penetrant in ro or sca null
mutants or in any of the ed mutants
(Fig. 3A-C). Therefore none of
these genes are absolutely required for R8 resolution. To test for redundancy,
we analysed double mutants ed4.12; roX63 and
ed4.12 scaBP2. These exhibit an increase in R8
twinning but in neither case is twinning fully penetrant
(Fig. 3D,E; data not
shown).
|
Egfr signalling is responsible for R8 twinning in ed
mutants
R8 resolution requires communication within the equivalence group, and R8
twinning is therefore a failure in this communication. It might be presumed
that R8 twinning results from a defect in Notch-mediated lateral inhibition,
as is likely for sca (Powell et
al., 2001). We did not find any evidence for this. The
ed4.12 R8 twinning phenotype is not altered by loss of one
copy of the Notch null allele, N55e11
(Fig. 4A). Moreover we observed
no change in expression in the morphogenetic furrow of the Notch target genes
of the E(spl) complex, as detected by antibodies that recognise
multiple family members (Ligoxygakis et
al., 1998
) (Fig.
5A,B) or by in situ hybridisation for E(spl)m8 mRNA (data
not shown). This suggests that Notch signalling is not the primary target of
ed. In searching for other pathways that may be affected, we found
that the R8 twinning phenotype of ed4.12 is strongly
suppressed by removing one copy of the Egfr gene
(EgfrIK35/+) (Fig.
4A-C) or of the Ras1 gene (data not shown). Conversely,
removing one copy of argos, which encodes an Egfr antagonist,
strongly enhances the R8 twinning phenotype of ed4.12
(Fig. 4A). This is particularly
striking because null mutations of argos exhibit no R8 twinning
phenotype (Baonza et al., 2001
;
Yang and Baker, 2001
). These
data suggest that ed may encode an Egfr antagonist that functions
during R8 specification.
|
|
In contrast to ed, R8 twinning in sca mutants is
apparently not caused by Egfr derepression. Examining R8 twinning in
EgfrIK35 sca double mutant clones is difficult because of
their combined IG spacing defects (Baonza
et al., 2001). However, we can unambiguously observe twinned R8s
in such clones (Fig. 4G). Given
the strong link between sca and the Notch signalling pathway
(Baker and Zitron, 1995
;
Powell et al., 2001
), it is
likely that R8 twinning in sca mutants is mediated by disruption of
Notch signalling. This finding reinforces the significance and specificity of
Egfr involvement in the ed twinning phenotype. It also demonstrates
that R8 twinning can be caused by at least two different mechanisms, which are
differentially affected in ed and sca mutants. This would
explain the lack of strong interactions between ed and
sca.
Egfr signalling is hyperactivated in ed mutants
Our data indicate that Egfr inhibition by ed is required for
correct R8 resolution. To see how Egfr signalling may be affected by
ed, we examined the expression of pointed-P1
(pnt-P1) mRNA and of the phosphorylated form of the Erk MAP kinase
(dpErk) (Gabay et al., 1996;
Gabay et al., 1997
). The
pattern and level of each reflects a direct response to Egfr activation.
Interestingly, in wild-type eye discs, dpErk
(Kumar et al., 1998
;
Wasserman et al., 2000
) and
pnt-P1 mRNA are both detectable in the IGs and R8 equivalence groups,
indicating that Egfr signalling is active in these locations
(Fig. 5C,E). Clearly, such
signalling does not normally interfere with R8 equivalence group resolution;
it may mediate a proposed function of Egfr signalling during IG spacing
(Dominguez et al., 1998
;
Spencer et al., 1998
;
Chen and Chien, 1999
;
Lesokhin et al., 1999
;
Yang and Baker, 2001
). In
ed mutant eye discs, the patterns of pnt-P1 and dpErk are
unchanged, but the levels of both are elevated in the IGs and equivalence
groups (Fig. 5D,F). Of the
ed mutations analysed (ed4.12,
edlH23/Df(2L)ed-dp, and l(2)k01102/Df(2L)ed-dp),
this effect is most noticeable for ed4.12, thereby
correlating with the higher incidence of R8 twinning observed for this allele.
This suggests that ed inhibits the level of Egfr signalling rather
than the pattern, and that this is normally sufficient to prevent such
signalling from interfering with R8 resolution. It also suggests that
ed antagonises Egfr signalling upstream of Erk activation.
Given these findings, we asked whether experimental Egfr pathway activation
might mimic ed mutation and provoke R8 fates. Interestingly, we found
evidence that this is the case if we drive expression of downstream components
of the pathway. Thus, when UAS-pnt-P1 or
UAS-RafAct was expressed in the eye posterior to the
morphogenetic furrow using a GMR-Gal4 driver, we could detect frequent
instances of twinned sens-expressing cells. Some of these twins
co-express ato, although more posteriorly than normal
(Fig. 4H, and data not shown).
These data suggest that the inhibitory function of ed can be bypassed
by expression of these components, implying that ed functions
upstream of Raf. This twinning phenotype, however, could not be
reproduced by identical misexpression of UAS-sSpi, the activated form
of the Spi ligand (data not shown). We showed earlier that Spi is not the
ligand responsible for R8 twinning, but if we assume that UAS-sSpi is
otherwise able to act in this situation as it can in other Egfr-mediated
processes in the eye (Freeman,
1996), then these data suggest that ed cannot be bypassed
by increased ligand and that ed therefore acts downstream of ligand
function. These findings, and the membrane associated nature of the Ed
protein, support a model in which Ed interacts directly with Egfr or a closely
associated component. Consistent with this, Ed protein is found at the apical
cell surface with Egfr (E.L.R., N.M.W. and A.P.J., unpublished).
ed is required within the equivalence group to prevent R8
twinning
Clonal analysis was used to explore whether ed prevents Egfr
signalling to prospective R8 cells or prevents R8 cells from receiving or
responding to the signal. We generated edlH23,
ed6.1 and ed4.4 mutant clones in eye
discs and examined them for Ato, Sens and Boss expression. Where wild-type and
mutant tissue was juxtaposed, R8 twins could frequently straddle the border,
being composed of one wild-type and one mutant R8 cell
(Fig. 6A, arrow). These
presumably represent cases where an equivalence group was bisected by the
clone border, and are consistent with ed acting both autonomously and
non-autonomously. We also observed rare cases of R8 twins consisting of two
genetically wild-type cells immediately juxtaposed to mutant tissue (five
cases out of 45 clones examined) (Fig.
6B, arrowhead). No R8 twins consisting of wild-type cells were
ever observed elsewhere in these discs. This can be explained if the third
member of an equivalence group was mutant for ed but did not
differentiate as an R8, therefore suggesting that ed acts
non-autonomously in this case. Moreover, we suggest that the low frequency of
this class of twins implies that the ed-associated signalling events
are occurring within the equivalence group rather than between the equivalence
group and surrounding cells. In other words, the phenotype is only seen on the
rare occasions when an equivalence group is bisected by the clone but the
mutant cell does not become an R8.
|
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DISCUSSION |
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The finding that Egfr signalling can induce R8 specification even
though it does not normally do so may resolve the contradictory evidence for
Egfr function in R8 specification. Recent studies definitively show that R8
cells can be specified in the absence of Egfr, albeit aberrantly
(Baonza et al., 2001;
Kumar et al., 1998
;
Yang and Baker, 2001
). Yet
Spencer et al. (Spencer et al.,
1998
) presented data that strongly suggested a link between R8
selection (not just IG spacing) and Egfr/Ras signalling. They observed that
expression of activated Ras results in strong ato upregulation and
ectopic R8 cells and that argos misexpression inhibits R8 formation
(Spencer et al., 1998
). The
latter findings may allude not to an Egfr requirement during R8 selection, but
to the ability of aberrant Egfr signalling to induce R8s.
Bai et al. (Bai et al.,
2001) suggested that ed acts downstream of the Egfr
target gene pnt-P1 in R7 specification and based on this they
proposed a hypothetical parallel signalling pathway that antagonises Egfr. Our
observations are more consistent with membrane-associated Ed interacting
directly with Egfr or with immediate downstream components. We observed
increased activated MAPK and pnt-P1 expression in ed
mutants, which suggests that ed acts upstream of MAPK activation in
the Egfr signalling pathway. Moreover, forced expression of pnt-P1 or
activated Raf can bypass the inhibitory function of ed,
whereas spi cannot. This is entirely consistent with the finding that
Ed is colocalised with Egfr at the cell surface (E.L.R., N.M.W. and A.P.J.,
unpublished) and that Ed can bind Egfr protein and is phosphorylated in
response to Egfr activation (Spencer and
Cagan, 2003
). Moreover, these findings are consistent with known
features of the L1 family of cell adhesion molecules (CAMs), with which Ed
protein shares extensive homology in its extracellular portion
(Bai et al., 2001
). L1 CAMs are
involved in the control of axon outgrowth, where they are associated with
regulation of Fgfr and Egfr activity
(Williams et al., 1994
;
Schaefer et al., 1999
;
Kamiguchi and Lemmon, 2000
;
Garcia-Alonso et al., 2000
).
In brain extracts, L1 physically associates with the MAPK cascade components
Raf1 and Erk2, while in vitro Erk2 can phosphorylate the L1 cytoplasmic domain
(Schaefer et al., 1999
).
Interestingly, our clonal analysis suggests both autonomy and nonautonomy,
suggesting that Ed might be able to interact with Egfr in trans as well as in
cis. If so, this might imply an association between the extracellular domains
of the two proteins. The molecular mechanism of L1 function is unclear,
although its endocytosis may be important for downstream events
(Schmid et al., 2000
). This
may have implications for Ed function. However, the intracellular domain of Ed
is distinct from that of L1 and there is evidence that tyrosine
phosphorylation within this domain is important for function, and that Ed may
act on Egfr via an interaction with the phosphatase encoded by
corkscrew (Spencer and Cagan,
2003
).
Unlike negative regulators such as argos, mutation of ed
does not alter the pattern of Egfr activation, just the intensity, suggesting
that the function of ed is to limit the level or duration of
activation. In support of this, Spencer and Cagan
(Spencer and Cagan, 2003)
provide biochemical evidence that the inhibitory activity of Ed is dependent
post-translationally on Egfr signalling, thereby providing a negative feedback
mechanism to damp down Egfr signalling. ed does not completely
suppress Egfr signalling around the morphogenetic furrow, presumably because
such signalling has some role to play. Indeed this wild-type level of
signalling may be important for mediating the proposed inhibitory Egfr/Ras/Raf
process in which one row of IGs helps to pattern the next row
(Chen and Chien, 1999
;
Baonza et al., 2001
;
Yang and Baker, 2001
)
(Fig. 7). Such activity occurs
at the same time that R8 fate must be restricted within the IGs by lateral
inhibition. Given the inductive nature of Egfr signalling generally, such
signalling could therefore interfere with R8 resolution. Therefore, in R8
proneural clusters ed must suppress a potential outcome of Egfr
signalling in the morphogenetic furrow (induction of R8 fate) rather than the
signalling itself.
|
Why does Egfr signalling induce R8 fate in ed mutants? It may
reflect the general inductive ability of Egfr in the context of cells primed
to become R8s. An alternative, however, is suggested by the close relationship
between Egfr and ato function. The wild-type level of Egfr signalling
in the morphogenetic furrow is dependent on ato
(Chen and Chien, 1999).
Moreover, increased ato expression in R8 precursors can provoke R8
twinning in a non-autonomous manner (White
and Jarman, 2000
), presumably by hyperactivation of Egfr
signalling. This relationship between ato and Egfr is reminiscent of
the normal function of ato during chordotonal SOP selection. In the
femoral chordotonal organ, ato triggers SOP recruitment by activating
Egfr signalling (zur Lage and Jarman,
1999
). In turn, Egfr signalling activates ato and SOP
fate in uncommitted cells in a manner that is suggestive of the aberrant
effect of Egfr on R8 specification in ed mutants. We speculate
therefore that R8 twinning might be an aberrant outcome of an
ato-Egfr neural recruitment network in the wrong time and place. It
is notable that chordotonal recruitment is unaffected in ed mutants
(E.L.R., N.M.W. and A.P.J., unpublished). Thus, by modulating Egfr signalling
specifically in the eye, ed enables the ato-Egfr network to
be customised to the specific needs of R8 precursor patterning, where Egfr
signalling must be activated by ato but supernumerary R8
specification must be prevented (Fig.
7). A key principle of development is the continual redeployment
of a handful of intercellular signalling pathways such as Egfr. As such, much
of development must involve similar instances of suppression of potential
developmental outcomes that would result from the re-use of signalling
networks.
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ACKNOWLEDGMENTS |
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