MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
* Author for correspondence (e-mail: MF1{at}mrc-lmb.cam.ac.uk)
Accepted 5 August 2003
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SUMMARY |
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Key words: Eye, Patterning, Ommatidial rotation, Adhesion, Epidermal growth factor receptor, spitz, keren, scabrous, nemo, roulette, argos
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Introduction |
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Eye differentiation begins in the third larval instar, when the
morphogenetic furrow sweeps across the eye imaginal disc from posterior to
anterior, leaving clusters of differentiating cells behind it (reviewed by
Wolff and Ready, 1993;
Ready et al., 1976
). The R8
photoreceptor cell is the first to be specified, followed sequentially by the
other photoreceptors in a defined order
(Tomlinson, 1985
;
Tomlinson and Ready, 1987
).
Non-neuronal cone and pigment cells are subsequently recruited to make up the
complete ommatidium. As well as this posterior to anterior organisation, the
eye disc is also polarised in the dorsoventral axis. The rhabdomeres of the
adult photoreceptors are arranged in a trapezoidal shape, and ommatidia in
dorsal and ventral halves of the disc are of opposite chiral forms, being
mirror-symmetric about the equator. This asymmetry arises in the third instar
disc, when the R3 and R4 cell fates are specified from an equivalent pair of
neuronal cells: the cell closest to the equator becomes R3, and the more polar
cell differentiates as R4. Subsequently, the ommatidia initiate rotation in
opposite directions on either side of the equator. Determination of chirality
is under the control of planar cell polarity (PCP) (reviewed by
Adler, 2002
;
Mlodzik, 1999
;
Strutt and Strutt, 1999
).
Mutations in PCP components, such as the Wnt receptor Frizzled, display
defects in chirality, with R3 and R4 being incorrectly specified, and rotation
being initiated in the wrong direction
(Zheng et al., 1995
).
Consequently, no equator is visible in a frizzled
eye.
In addition to determination of chirality and direction of rotation, the
developing ommatidial clusters must also rotate by the correct degree. Visual
processing in flies involves a precise mapping of the pattern of
photoreceptors onto neurons in the optic lobe
(Clandinin and Zipursky, 2002;
Meinertzhagen and Hanson,
1993
). Therefore, if the ommatidia are not properly rotated, the
photoreceptor array will be disorganised, leading to a loss of visual acuity.
The precision of ommatidial rotation is remarkable and is easily seen in
sections through adult eyes (see, for example,
Fig. 1A). In the wild-type (WT)
disc, the ommatidia first rotate through 45°, then pause before
reinitiating rotation to complete the full 90° turn (shown schematically
in Fig. 1E). As well as
affecting chirality, frizzled mutants show disruptions in the degree
of rotation (Zheng et al.,
1995
), although this does not seem to be true for all PCP
components; mutations in the atypical cadherin fat, for example, show
significant chiral defects but ommatidia still rotate through 90°
(Rawls et al., 2002
;
Yang et al., 2002
). Chirality
and rotation, although intimately linked, are therefore separable processes,
with the control of rotation being much less well understood than
chirality.
|
Here, we identify a role for the Drosophila epidermal growth
factor receptor (Egfr) signalling pathway in the control of rotation. Egfr
signalling plays several important roles during eye development. Notably, it
is responsible for the recruitment of all cell types except R8 in the
developing ommatidium in the absence of Egfr signalling, only R8
differentiates, and overactivating the pathway leads to excess photoreceptor
and cone cell recruitment (Freeman,
1996; Freeman,
1997
). As well as its role in recruitment, this pathway also
controls ommatidial spacing, promotes cell proliferation behind the
morphogenetic furrow, and protects cells against apoptosis
(Baker and Yu, 2001
;
Baonza et al., 2001
;
Bergmann et al., 1998
;
Domínguez et al., 1998
;
Kumar et al., 1998
;
Kurada and White, 1998
;
Spencer et al., 1998
).
Both over- and underactivation of the Egfr signalling pathway gave similar rotational defects in the adult eye. To our surprise, however, we found that the initial process of ommatidial rotation is not dependent on Egfr signalling. Instead, the rotational angle becomes disrupted at a later stage in development, suggesting that this pathway may be required to prevent ommatidia from reinitiating rotation during pupal stages, or to protect them against rotational distortion during the substantial morphogenetic movements that occur during the formation of the mature retina. These results demonstrate a previously unrecognised additional role for Egfr signalling in eye formation, further emphasising the reiterative functions of a single signalling pathway in development. Moreover, they provide us with the opportunity of using the well-characterised system of the fly eye to analyse mechanisms of regulating cell motility and tissue remodelling. As an initial step in this direction, we have evidence that ommatidial rotation may depend at least partly on cadherin-based adhesion.
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Materials and methods |
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Histology
Adult heads were embedded as described in Freeman et al.
(Freeman et al., 1992). Larval
eye discs and pupal retinae were stained as described
(Gaul et al., 1992
). The
following antibodies were used: mouse anti-ß-galactosidase (1:100)
(Promega), rabbit anti-ß-galactosidase (1:100) (Cappel); mouse anti-Cut
(1:100) (Blochlinger et al.,
1990
) and rat anti-Elav (1:200)
(O'Neill et al., 1994
) (both
obtained from DSHB); and rabbit anti-BarH1 (1:50)
(Higashijima et al., 1992
).
Alexa-568 and Alexa-647 (Molecular Probes) and FITC-conjugated secondary
antibodies were used at 1:200 (Jackson ImmunoResearch). Fluorescent images
were taken on a BioRad Radiance confocal microscope.
Analysis of rotational angles
In those cases in which rotational angles were measured accurately, this
was done using the program XIMDISP (Smith,
1999). Adult eye sections were photographed and images imported
into XIMDISP. The angle calculated was that between a vector drawn along the
equator and a vector from rhabdomeres R1-R3. All correctly specified ommatidia
in a section were analysed. In all other cases, ommatidia in adult eye
sections or larval discs were scored as being misrecruited, correctly
orientated or misrotated. Frequencies of each type were then calculated.
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Results |
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Further examination of the adult phenotype indicated that it is rotation
specifically that is disrupted on overexpressing Keren; the chirality (i.e.
the correct specification of R3 and R4) of the ommatidia remains unaffected.
This distinguished the UAS-keren phenotype from disruption of PCP
components, which can cause both rotational and chiral defects
(Theisen et al., 1994;
Zheng et al., 1995
).
Rotational defects are caused by disrupting Egfr signalling
Keren resembles the Egfr ligands Spitz and Gurken and can activate the Egfr
(Reich and Shilo, 2002;
Urban et al., 2002
). The
absence of a keren mutant, however, prevents us from being sure that
it does not act in another pathway. The unexpected rotational disruption could
be explained either by a previously unrecognised function of the Egfr, or by
Keren acting through a different mechanism. Analysis of a role for Egfr
signalling in rotation is made difficult by the fact that this pathway plays
many roles in eye development. For example, disrupting signalling usually
affects photoreceptor recruitment, making ommatidial rotation unscorable. We
therefore examined another condition in which the Egfr pathway is only
moderately hyper-activated: a hypomorphic mutation in the Egfr inhibitor
argos (Schweitzer et al.,
1995
) (Fig. 2A,B).
In argosw11 clones, although many of the ommatidia had too
many photoreceptors (circles), a significant proportion had the correct number
and we observed that many of these ommatidia were misrotated. This implies
that the rotation phenotype caused by misexpressing Keren is a consequence of
overactivating the Egfr pathway, rather than being a non-Egfrrelated function
of Keren. We note that these data do not address whether Keren normally
functions in ommatidial rotation; instead they simply demonstrate that Egfr
hyperactivity including that triggered by Keren leads to
misrotation. Below we consider the possible ligands involved.
|
All known cases of Egfr signalling in Drosophila are transmitted
through the canonical Ras/Raf/MAPK pathway, and through a transcriptional
output. The transcription factor Pointed is involved in most circumstances:
PointedP2 is directly phosphorylated and activated by MAPK, and upregulates
the expression of PointedP1; both factors mediate the transcription of
downstream genes (Brunner et al.,
1994; Klämbt,
1993
; O'Neill et al.,
1994
). In the case of rotation, which we envisage as being a
specialised case of cell motility or tissue remodelling, it seemed possible
that Egfr signalling might influence the cytoskeleton directly, rather than
exerting its effects by transcriptional control. We therefore tested whether a
pointed hypomorph showed rotational defects
(Fig. 2I,J). Although, as
expected, many ommatidia showed under-recruitment of photoreceptors,
rotational defects were frequent in those ommatidia that were correctly
specified, indicating that this function of the Egfr pathway relies on
Pointedmediated transcription.
The rotational phenotypes caused by perturbation of Egfr signalling were
very similar to the published phenotype of the roulette mutation, one
of the few mutations previously reported to specifically disrupt rotation and
not chirality (Choi and Benzer,
1994). Interestingly, roulette turns out to be allelic to
argos (K. Choi, personal communication). We confirmed this by
non-complementation of roulette by argosw11, and
by rescue of the roulette phenotype by a sev-argos transgene
(Fig. 2L-Q). This result is
therefore consistent with our discovery of a role for the Egfr pathway in
controlling ommatidial rotation. We hereafter refer to the roulette
mutations as argosrlt.
More than one Egfr ligand controls rotation
There are four ligands that activate the Drosophila Egfr: Spitz,
Gurken and Keren, which resemble mammalian TGF, and Vein, a
neuregulin-like molecule
(Neuman-Silberberg and Schüpbach,
1993
; Reich and Shilo,
2002
; Rutledge et al.,
1992
; Schnepp et al.,
1996
; Urban et al.,
2002
). Spitz is thought to mediate most of the Egfr functions in
eye development (Freeman,
1994
; Freeman,
1997
; Tio et al.,
1994
; Tio and Moses,
1997
), although spitz clones do not phenocopy
Egfr clones in all respects. Specifically, spitz clones do
not show defects in cell survival or ommatidial spacing, which are seen in
Egfr loss-of-function clones
(Domínguez et al.,
1998
). We examined spitz hypomorphic eyes to determine
whether these show rotational defects (Fig.
3A,B). Under-recruited ommatidia are very common in the
spiscp1 hypomorph, indicating that Egfr activity is
substantially impaired to beneath the threshold for photoreceptor
recruitment. Despite this, very few misrotated ommatidia are seen (see
Fig. 3C). In comparison,
ru1 eyes show only minor recruitment defects, indicating a
less dramatic reduction of Egfr activity than spiscp1.
ru1 eyes, however, show severe rotational defects. These data
suggest that Spitz is not essential for normal rotation. They do not, however,
rule out the possibility that Spitz acts redundantly with another ligand. To
test this, we looked for a genetic interaction between Star and a
spitz hypomorph (see Fig.
3C). As expected, heterozygosity for spitz enhanced the
recruitment defects in the S/+ eye. We also observed a significant
enhancement of rotational defects, implying that Spitz does function in
ommatidial orientation. Together, these results suggest that Spitz acts
redundantly with another Egfr ligand to control rotation. The fact that loss
of Rho3/ru, a protease that activates Egfr ligands
(Wasserman et al., 2000
),
results in rotational defects, whereas spitz mutants do not, implies
the involvement of another cleaved ligand. Gurken is restricted to the
germline. By elimination, we therefore tentatively conclude that Keren also
acts in the Egfr-dependent regulation of ommatidial rotation. Note, however,
that keren expression is too low to detect by in situ hybridisation
in any tissue (Reich and Shilo,
2002
) (K.E.B. and M.F., unpublished) so we cannot tell whether it
is transcribed appropriately. Confirmation of our hypothesis awaits the
identification of a keren mutant.
|
Initial rotation is unaffected by the Egfr
At what stage in ommatidial development does the Egfr control rotation? We
used several markers to look at rotation in the eye disc: -Bar, which
stains R1 and R6 (Higashijima et al.,
1992
), svp-lacZ, which is strongly expressed in R3 and R4
and more weakly in R1 and R6 (Mlodzik et
al., 1990b
), and m
0.5-lacZ, which
highlights R4 only (Cooper and Bray,
1999
). The first two markers enable visualisation of the
rotational angle during disc development, and the third shows which cell of
the R3/R4 pair develops R4 fate, thus providing a marker for chirality.
m
0.5-lacZ staining of discs misexpressing
keren showed no defects in R3/R4 specification (compare
Fig. 4A and
Fig. 4C), which correlates well
with the lack of chiral defects in the adults. Surprisingly, rotational
defects were also very minor in the third instar disc
(Fig. 4E-L). The vast majority
of ommatidia reach 45° as expected, and by the back of the disc have
turned to 90°. This is in stark contrast to the adult eye, in which
approximately 28% have rotated less than 90°, and 6.2% less than 45°,
as well as 65% being rotated greater than 90°. Occasional misrotations can
be seen in the larval disc (arrowheads in
Fig. 4E,G), but analysis showed
that the frequency of these (4.9%; 1275 ommatidia in 10 discs) is not
significantly different from WT (5.1%; 1354 ommatidia in 8 discs). This result
demonstrates that the eye defects we see in the adult must arise at a stage
later in development than the third instar imaginal disc.
|
|
|
Genetic interactions with other known rotation mutants
Apart from Argos and other members of the Egfr pathway, Nemo and Scabrous
are the main factors known to cause rotation-specific defects
(Choi and Benzer, 1994;
Chou and Chien, 2002
). We
therefore tested potential genetic interactions between the Egfr pathway,
nemo and scabrous. It is already known that nemo,
argosrlt double mutants show a nemo phenotype
(Choi and Benzer, 1994
); this
was also observed on misexpressing keren in a
nemoP1 mutant background (compare
Fig. 6A,B with
Fig. 6C,D). In addition,
ru1 nemoP1 double mutants were
indistinguishable from the nemoP1 single mutant
(Fig. 6E,F), implying that
there is no synergy between the Egfr pathway and nemo. In conjunction
with the observations that Nemo is required for the onset of the second
45° rotation, whereas Egfr activity is not required until later, this
suggests that they act in separate processes. Moreover, these data imply that
Egfr activity is not required unless ommatidia rotate beyond the initial
45°.
|
Cadherin-based adhesion is involved in rotation
Our results demonstrate that Egfr signalling is required for the
maintenance through eye development of the correct orientation of ommatidia.
We speculated that rotation may rely at least partly on the adhesive
properties of the cells. In an initial attempt to examine this hypothesis, we
looked for genetic interactions between components of the Egfr pathway and
various adhesion molecules. We used a Star heterozygote, in which
Egfr signalling is slightly reduced
(Kolodkin et al., 1994), as a
background in which to look for interactions, because this phenotype is very
weak (see Fig. 2C,D), allowing
any enhancement of rotational defects to be easily recognised. Halving the
dose of
-laminin [wing blister
(Martin et al., 1999
)] and the
integrin ß subunit [myospheroid
(MacKrell et al., 1988
)] did
not modify the Star/+ phenotype. In contrast, alleles of E-cadherin
[shotgun (Tepass et al.,
1996
)] showed a significant interaction with Star, with
many more misrotated ommatidia (Fig.
7). Under the strongest condition, there was also an enhancement
of the rare misrecruitment defects seen in Star/+ eyes, but the
enhancement of the rotational defect was independent of this by two criteria.
First, the rotational defects were only measured in correctly specified
ommatidia; and second, the weaker alleles of shotgun affected
rotation without enhancing recruitment. On the basis of these results, we
conclude that the control of rotation by Egfr signalling is linked to
cadherin-based adhesion.
|
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Discussion |
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Egfr signalling is already known to play several important roles in eye
development, including cell recruitment, ommatidial spacing, cell
proliferation and survival (Baker and Yu,
2001; Baonza et al.,
2001
; Bergmann et al.,
1998
; Domínguez et al.,
1998
; Freeman,
1996
; Kumar et al.,
1998
; Kurada and White,
1998
; Spencer et al.,
1998
). The identification of a further function in
ommatidial rotation emphasises the pleiotropic effects of one
signalling pathway in the development of a single tissue, and highlights the
question of how such diverse successive effects are coordinated. One answer is
that the signal itself does not specify the cellular consequences. Instead it
is the developmental state of the receiving cell mechanistically, its
repertoire of signal-responsive transcription factors that determines
the outcome of signalling (Flores et al.,
2000
; Freeman,
1997
; Xu et al.,
2000
). We suspect that another important factor in regulating
reiterative signalling in the eye is the use of two different activating
ligands: Spitz, which triggers cell recruitment and mitosis
(Baker and Yu, 2001
;
Domínguez et al., 1998
;
Freeman, 1996
;
Tio and Moses, 1997
), and
Keren, which is inferred to control ommatidial spacing and survival
(Wasserman et al., 2000
), and
which we hypothesise here to participate in rotational control. Importantly,
however, there is no evidence that the different ligands produce different
`qualities' of signal; on the contrary, all current results support the idea
that the ligands activate exactly the same effector pathways (see
Gabay et al., 1997
). Rather,
we imagine that multiple activating ligands could allow for a more precise and
complex regulation of the initiation of signalling. These important issues,
however, will only be fully resolvable when keren mutants are
isolated.
The fact that over- or underactivating the Egfr pathway has similar effects
on rotation indicates that it is either the precise levels of signalling or
the spatial distribution of signal activation that is important for
controlling orientation. In the latter hypothesis, correct ommatidial rotation
depends on asymmetric Egfr signalling in a specific subset of cells within
each ommatidium. Therefore, global hyperactivation or loss of signalling would
have similar effects because both conditions would disrupt the asymmetry
required for function. The planar polarity receptor Frizzled shows this kind
of dependency on asymmetric activation loss and gain of Frizzled
function in the eye show the same type of defects
(Strutt et al., 1997;
Tomlinson et al., 1997
),
because Frizzled must be preferentially active in R3 but not R4 in order to
exert its effects (Cooper and Bray,
1999
; Fanto and Mlodzik,
1999
; Tomlinson and Struhl,
1999
).
Perturbing Egfr signalling appears to be different from all other known
rotation mutants, in that it exerts its effects on rotation after the normal
process has been completed. In nemo and scabrous mutants,
defects can be seen in the disc, while the ommatidia are still rotating
(Choi and Benzer, 1994;
Chou and Chien, 2002
). This is
also the case for Frizzled and other PCP components, which affect rotation at
early stages (Zheng et al.,
1995
). Conversely, under conditions in which Egfr signalling is
disrupted, ommatidia rotate and stop rotating precisely as they should, and
yet the adult eyes show significant defects in ommatidial orientation. These
observations imply that Egfr signalling is acting in a distinct process from
other known components, that of maintaining ommatidial orientation after
rotation is complete. Despite this evidence for a new aspect of rotational
control affecting pupal eye development, our data show that Egfr signalling is
actually required during the third larval instar, during or immediately after
the second 45° rotation if the pathway is disrupted at this time,
rotational defects are seen in the adult eye. It would appear, therefore, that
there is a delay between the time at which Egfr signalling is required and the
time at which the phenotype becomes apparent.
A model that might account for these results is that the role of Egfr signalling is to establish a `locking' mechanism that ensures that ommatidia remain in their final orientation. Such a mechanism might be necessary to protect the ommatidia against positional disruption during later events in eye development. Signalling would therefore be required during or at the end of normal rotation in order to set in place this hypothetical `lock', although defects might not arise until significantly later than this, when processes occur that would cause ommatidia to reorientate in the absence of such a lock.
What might such processes be? During pupal development, the eye undergoes
significant changes (Cagan and Ready,
1989; Wolff and Ready,
1993
) (see Fig. 8).
Additional cell types primary, secondary and tertiary pigment cells
are recruited into the ommatidium from approximately 12 hours
post-pupariation. Also at about this time, the eye disc everts, an event
involving significant morphogenetic movement. Later, there is a phase of
apoptosis starting at approximately 24 hours of pupal life, which is preceded
by a reorganisation of interommatidial cells into a tight lattice network
surrounding each cluster (Cagan and Ready,
1989
). Later still, a further stress on the tissue might be
rhabdomere morphogenesis, which initiates at approximately 37 hours and
involves substantial cellular gymnastics
(Longley and Ready, 1995
). Any
of these events could result in morphogenetic stresses on the eye tissue that
could disrupt the precise rotational organisation of ommatidia. In this model,
the presence of an Egfr-controlled lock functions to prevent such rotational
disruption. The fact that loss of Egfr signalling has no effect in
nemo mutant ommatidia might imply that there is a Nemo-dependent
change in the adhesive properties of the cells when ommatidia commence the
second 45° rotation; before this point, they are not sensitive to later
disruption. The observation that shotgun mutants specifically enhance
rotational defects of Star heterozygous eyes is consistent with this
kind of model: Egfr signalling would result in a change of the adhesive
properties of the cells, thereby restricting their motility with respect to
their neighbours. Significantly, E-cadherin and Egfr signalling are associated
in several other morphogenetic processes in Drosophila development
(Dumstrei et al., 2002
;
Fulga and Rorth, 2002
;
James et al., 2002
).
|
The Drosophila eye provides a striking example of the precision
with which developmental patterning can occur. Ommatidia in the adult eye are
precisely orientated in essentially 100% of cases. Our results suggest that
this precision is not simply a consequence of an initial rotational process,
but also critically depends on at least two further aspects. First, there is
an Egfr-dependent mechanism protecting the eye against disruption of the
original pattern presumably caused by the morphogenetic and cellular upheavals
that occur in pupal stages. Second, we have evidence for a refinement and
error-correcting mechanism, whose molecular basis is unknown. Another aspect
of fly eye patterning, cell recruitment, also depends on a two-stage process
of initial patterning followed by refinement. In this case, too many cells are
originally produced, presumably to ensure there are enough to form all
necessary cell types. This is followed by specific apoptotic removal of
superfluous cells in pupal life (Cagan and
Ready, 1989; Miller and Cagan,
1998
; Wolff and Ready,
1991
). We suspect that it may prove to be a general property of
pattern formation especially when great precision is required, as in
the case of a visual system that refinement and active maintenance
functions are programmed into the overall patterning process.
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ACKNOWLEDGMENTS |
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