Development 121, 4247-4256 (1995)
Printed in Great Britain © The Company of Biologists Limited 1995
DEV8284
4247
Ommatidial polarity in the Drosophila eye is determined by the direction of
furrow progression and local interactions
David I. Strutt and Marek Mlodzik
Differentiation Programme, EMBL, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
SUMMARY
The adult eye of Drosophila is a highly ordered structure.
It is composed of about 800 ommatidia, each displaying
precise polarity. The ommatidia are arranged about an axis
of mirror image symmetry, the equator, which lies along
the dorsoventral midline of the eye. We use hedgehog
pathway mutants to induce ectopic morphogenetic furrows
and use these as a tool to investigate the establishment of
ommatidial polarity. Our results show that ommatidial
clusters are self-organising units whose polarity in one axis
is determined by the direction of furrow progression, and
which can independently define the position of an equator
without reference to the global coordinates of the eye disc.
INTRODUCTION
different terms, relative to the direction of furrow progression
and on which side of the equator they lie (summarised in Fig.
1).
Little is known of the mechanisms underlying ommatidial
rotation and chirality. However, the known mutations disrupting the process affect ommatidia as discrete units, the individual ommatidia themselves being correctly assembled. This
suggests that assembly is independent from the establishment
of polarity. In the nemo mutant, ommatidia initially rotate 45
degrees, but then fail to complete rotation to 90 degrees, whilst
in the roulette mutant ommatidia rotate further than 90 degrees
(Choi and Benzer, 1994). As chirality and orientation relative
to the equator remain normal in these two mutations, they
appear to be involved in the actual mechanics of rotation, or in
the reception or interpretation of signals required to determine
the degree of rotation, rather than in the establishment of
polarity. A second class of genes, the tissue polarity mutants
(Gubb and García-Bellido, 1982), affect ommatidial polarity,
producing defects in both rotation and chirality (Gubb, 1993;
Theisen et al., 1994). These genes are good candidates for
being involved in a signalling pathway required to transmit
polarity information. In particular, one of them (frizzled)
encodes a protein with structural similarity to a G-protein
linked receptor (Vinson and Adler, 1987; Vinson et al., 1989).
A major question is whether ommatidia respond to global
signals (throughout the disc epithelium) or to local signals
(e.g. between individual ommatidia), or both, when determining their polarity. A number of different models can be
envisaged for how correct ommatidial polarity could be
achieved. In an extreme ‘global coordinate’ model, each individual ommatidial cluster would determine its position
relative to global positional information present in the eye
disc, and then adopt the appropriate degree of rotation (in the
wild-type eye, always 90 degrees) and chirality. In fact, as
In the Drosophila eye imaginal disc, pattern formation and
associated differentiation begin during the third larval instar
stage, and proceed in a wave starting at the posterior edge of
the disc and progressing anteriorly. This wave is marked by
the passage of the morphogenetic furrow, an indentation in the
disc epithelium produced by transient cell shape changes. The
furrow takes about 2 days to traverse from posterior to anterior.
Ahead of it (anteriorly), cells are unpatterned and freely
dividing, whilst in and behind it (posteriorly) cells are recruited
into clusters of photoreceptors in a precise sequence, ultimately
giving rise to the geometrically arranged mature ommatidia
that constitute the adult eye (Ready et al., 1976; Tomlinson and
Ready, 1987; Wolff and Ready, 1991).
As the furrow advances in the anteroposterior axis, nascent
ommatidial clusters emerge in rows (row 0 being defined as
lying in the furrow itself). All clusters initially have a single
axis of symmetry and face in the same direction. However, by
row 6 the clusters have rotated by approximately 45 degrees
away from the anteroposterior axis. They maintain this angle
of rotation for about 10 rows, before rotating a further 45
degrees, bringing them 90 degrees from their original axis. The
clusters then remain in this orientation throughout the rest of
development and in the adult eye. Ommatidia in the dorsal and
ventral halves of the disc rotate in opposite directions. This
leads to the creation of an equator running along the dorsoventral midline, which corresponds to an axis of mirror-image
symmetry. Concommitant with their rotation, the ommatidial
clusters also lose their symmetry, and opposite chiral forms are
established in the dorsal and ventral halves of the eye (Dietrich,
1909; Tomlinson and Ready, 1987; Wolff and Ready, 1991).
Thus the final polarity of an ommatidium is defined relative to
both the dorsoventral and the anteroposterior axes, or in
Key words: Drosophila, furrow progression, tissue polarity,
ommatidial rotation, equator
4248 D. I. Strutt and M. Mlodzik
clusters are normally born with anteroposterior polarity (as a
result of the furrow moving in a defined direction), this model
can be simplified to postulate only the existence of a system
of positional information on the dorsoventral axis (Tomlinson,
1988; Baker and Rubin, 1992; Ma and Moses, 1995). A prediction of this model would be that the position of the equator
is fixed relative to the global coordinates of the disc. Alternatively, in a ‘self-organising’ model, ommatidial clusters would
determine their polarity by communicating with each other,
for instance in a manner that might be considered to be
analogous to crystallisation around a single nucleation centre
(Gubb, 1993). Such a nucleation centre would be provided by
an initial asymmetry, from which pattern would then
propagate outwards. Traditionally, models invoking positional
information have been favoured. For instance, classic grafting
experiments on the retina of the insect Oncopeltus suggested
that at least anteroposterior ommatidial polarity was determined by a gradient of positional information in the eye-disc
epithelium (Lawrence and Shelton, 1975).
In this study, we use mutations that induce ectopic morphogenetic furrows and neuronal differentiation to investigate the establishment of ommatidial polarity, and to test the
predictions of these models. Our results indicate that the final
polarity of ommatidia in the adult eye is dependent both
upon the direction of furrow progression and upon the ability
of ommatidial units to organise themselves independently to
form an equator. We find no evidence for the involvement
of a global positional information system in directly determining the polarity of ommatidia or the position of the
equator.
MATERIALS AND METHODS
Fly strains and generation of clones
The pka-C1 allele used was the lethal P-insertion pka-C1l(2)01272
(Lepage et al., 1995; Strutt et al., 1995). The ptc allele used was
ptc98/29, a lethal P-insertion identified in this laboratory, which
behaves as a strong allele by all criteria tested and has been confirmed
to carry no other mutations on the chromosome by reversion analysis
(D. S. and M. M., unpublished data). Disc clones of pka-C1l(2)01272
and ptc98/29 were marked using arm-lacZ reporters inserted at 28A and
51A, respectively (Vincent et al., 1994). dpp expression was
monitored using an insertion of the BS3.0 dpp-lacZ reporter gene on
the third chromosome (Blackman et al., 1991). The svpl(3)7842
enhancer trap allele was used to reveal svp expression (Mlodzik et al.,
1990).
Clones were generated using the FLP/FRT system (Xu and Rubin,
1993). For adult clones, larvae of genotype w hsFLP1; pka-C1l(2)01272,
P[ry+; hs-neo; FRT]40A / P[mini-w+; hs-πM]21C, 36F, P[ry+; hsneo; FRT]40A or w hsFLP1; P[ry+; hs-neo; FRT]43D, ptc98/29 /
P[ry+; hs-neo; FRT]43D were heat-shocked at 38˚C for 120 minutes
at 24-48 hours after egg-laying; clones were identified by the altered
dosage of the white mini-genes carried by the P[mini-w+; hs-πM] or
the ptc98/29 P-element insertions. For clones in discs marked by lack
of arm-lacZ expression and carrying the svp enhancer trap, larvae of
genotype w hsFLP1; pka-C1l(2)01272, P[ry+; hs-neo; FRT]40A / P[w+;
arm-lacZ]28A, P[ry+; hs-neo; FRT]40A; svpl(3)7842 / + or w hsFLP1;
P[ry+; hs-neo; FRT]43D, ptc98/29 / P[ry+; hs-neo; FRT]43D, P[w+;
arm-lacZ]51A; svpl(3)7842 / + were used. Hh misexpression was
carried out using flies of genotype y hsFLP1; P[ry+, Tub>y+>hh] / +
(Basler and Struhl, 1994), again heat-shocking for 120 minutes in the
first instar stage.
Histology
Antibody stainings of eye imaginal discs were carried out by standard
methods (Tomlinson and Ready, 1987). β-galactosidase staining and
immunohistochemical double staining was as previously described
(Strutt et al., 1995). Elav was detected using a rat monoclonal
antibody (gift of G. Rubin), and β-galactosidase was detected using
either a mouse monoclonal antibody (Promega) or a rabbit polyclonal
antibody (Cappell). Secondary antibodies conjugated to peroxidase,
FITC or Texas Red were used.
Standard histological methods were used for sections of adult eyes
(Tomlinson and Ready, 1987).
RESULTS
hh pathway mutations in the eye disc induce ectopic
morphogenetic furrows
Morphogenetic furrow progression is regulated by the
hedgehog (hh) signalling pathway (Heberlein et al., 1993; Ma
et al., 1993). Recently it has been shown that either misexpression of Hh protein or loss-of-function mutations in the
catalytic subunit of cAMP-dependent protein kinase A (pkaC1, another component of the pathway) can induce ectopic
expression of the TGF-β homologue decapentaplegic (dpp)
and ectopic morphogenetic furrows anteriorly to the endogenous furrow (Heberlein et al., 1995; Pan and Rubin, 1995; Strutt
et al., 1995).
The segment-polarity gene patched (ptc) (Nüsslein-Volhard
and Wieschaus, 1980) is also involved in the hh signalling
pathway in the embryo, wing and leg imaginal discs. ptc
encodes a multipass transmembrane protein, that acts downstream of hh, and has been proposed as a possible candidate
for the Hh receptor (Hooper and Scott, 1989; Nakano et al.,
1989; Ingham et al., 1991). In the anterior compartment of the
wing disc, ectopic dpp expression can be induced by either
ectopic Hh expression (Basler and Struhl, 1994), loss-offunction pka-C1 mutations (Jiang and Struhl, 1995; Lepage et
al., 1995; Li et al., 1995; Pan and Rubin, 1995), or loss-offunction ptc mutations (Capdevila et al., 1994).
Given the similarity of the pka-C1 and ptc mutant phenotypes
in the wing disc, it seemed likely that ptc clones in the eye disc
would also be capable of inducing ectopic morphogenetic
furrows, associated with ectopic dpp expression and neuronal
differentiation. This is indeed the case: in discs in which
homozygous mutant ptc clones have been induced, ectopic dpp
expression is seen anterior to the advancing furrow (Fig. 2A).
Close to the furrow itself, ectopic ommatidial clusters can be
seen surrounded by ectopic dpp expression (Fig. 2B). Thus as
for pka-C1 mutant clones, ptc clones are capable of inducing
ectopic photoreceptor differentiation and an associated outward
moving morphogenetic wave. Again, as for pka-C1, there is a
limited region in the zone anterior to the moving furrow that is
able to sustain such ectopic differentiation, which we refer to
as the ‘zone of competence’ (Strutt et al., 1995).
The ability to induce ectopic morphogenetic furrows
provides a powerful tool for investigating the relationship
between furrow progression and the establishment of pattern
and polarity in the eye disc. We have used Hh misexpression,
and loss-of-function clones of pka-C1 and ptc to analyse the
rotational behaviour of ommatidial clusters born from morphogenetic furrows that are not moving in the anteroposterior
axis.
Furrow progression and ommatidial polarity 4249
Direction of furrow progression provides the first
axis of polarity
We analysed first the initial orientation of ommatidial clusters
when they emerge from an ectopic furrow. In the wild-type
disc, the direction of furrow movement is always along the
anteroposterior axis. All ommatidia are then born lying in this
axis, pointing posteriorly. By inducing loss-of-function ptc or
pka-C1 clones, it is possible to produce furrows progressing in
any direction. In this case, the initial orientation of the clusters
is always along an axis coincident with the direction of furrow
progression (Fig. 3A).
The critical rôle of furrow movement in the initial determination of polarity can also be demonstrated in a second way:
when a dpp-expressing ptc or pka-C1 clone enters the ‘zone of
competence’ as a result of anterior furrow progression,
neuronal differentiation occurs within the clone, and ommatidial clusters are born de novo (at least in the posterior of the
clone), without emerging from a moving furrow. In many
cases, these ommatidia are missing photoreceptors, apparently
due to problems in recruitment caused by the nascent clusters
being irregularly spaced, making it difficult to assess their orientation. However, in ommatidia that are normally constructed,
orientation is found to be random. This can be seen both in
clones in discs (Fig. 3B), and then later in the adult after differentiation and patterning is complete (Fig. 3C). Whilst it is
possible that some normally constructed clusters are prevented
from rotating by being too closely positioned to their neighbours, we frequently see well-spaced normally constructed
clusters with random orientation. This suggests that clusters
that are not born from a moving furrow are unable to adopt a
correct orientation relative to either each other or their position
in the disc.
A second indication that clusters born within ptc or pka-C1
clones do not subsequently adopt polarity consistent with their
position within the disc is the presence of mixed chiral forms
of ommatidia in the adult eye. Normally all ommatidia in the
dorsal half of the eye have the same chiral form, which is the
mirror image of that adopted by ommatidia in the ventral part
of the eye (i.e. on the other side of the equator that runs along
the dorsoventral midline, see Fig. 1D). As both chiral forms
are seen within clones on either side of the equator (Figs 3C,
5A-C), we conclude that chirality cannot be determined by
absolute position within the disc.
Ommatidia born from ectopic furrows do not always
rotate towards the equator
Although ommatidia are normally born facing along the
anteroposterior axis, before photoreceptor recruitment is
complete they rotate 45 degrees towards the equator of the disc
(which runs along the dorsoventral midline) (Fig. 1B,C). Still
later, they turn a further 45 degrees, bringing them 90 degrees
from their original orientation, a configuration that is maintained in the adult eye (Fig. 1D). We have analysed the
rotational behaviour of ectopic ommatidial clusters born from
ectopic furrows progressing outwards from ptc and pka-C1
clones. In these cases, it is generally possible to predict the
initial orientation of the clusters (i.e. along an axis determined
by the direction of furrow progression), and thus to infer in
which direction they have rotated.
Clones were analysed in discs, using markers that permit the
Table 1. Summary of ptc clones* analysed by confocal
microscopy in discs
Total number of ptc clones examined†
Scorable ommatidial polarity defects‡
Fields of clockwise and anticlockwise rotated ommatidia
Opposing fields of rotation defining equators
41
29
12
10
*Consistent results were obtained for pka-C1 clones.
†Approximately 2000 discs were inspected by fluorescence microscopy, of
which 1 in 8 were the correct genotype. Discs with sufficiently large clones in
suitable positions that disrupted patterning as revealed by Elav staining were
selected for confocal imaging.
‡About 25% of the discs were not informative due to distortions in the
epithelium caused by overgrowth as often associated with ectopic dpp
expression (Strutt et al., 1995; Heberlein et al., 1995).
easy visualisation of the boundary of the clone and of ommatidial rotation, at a time when clusters would normally have
turned 45 degree towards the equator of the disc (Table 1). A
number of different observations were made (Fig. 4, see also
Fig. 6). Firstly, in clones on either the dorsal or the ventral side
of the equator, clusters are seen which rotate both clockwise
and counterclockwise, and either towards or away from the
equator of the disc. Thus, the direction in which clusters rotate
cannot be an intrinsic property of their position in the disc.
Moreover, neither is it obligatory that they rotate towards the
normal equator of the disc. Secondly, in some cases rotation
appears to be retarded, that is, clusters can be still facing along
the axis of inferred furrow progression, even though a cluster
of this maturity would normally have rotated 45 degrees.
Thirdly, we do not find clusters rotated more than 45 degrees
at this stage in their development. Thus, rotation does not
always occur at a fixed point in the maturational sequence of
ommatidial development, but if rotation has occurred then it is
limited in degree.
Ectopic ommatidia can organise themselves around
ectopic equators
Despite the fact that ectopic clusters do not always rotate
towards the equator, their organisation nevertheless does not
appear to be random. Instead, groups of clusters all rotating in
the same direction are often observed. Strikingly, when two
such groups of clusters are apposed, they can reveal the
presence of an ‘ectopic’ equator, i.e a line of symmetry about
which polarity is inverted.
As the degree of rotation around disc clones is limited, and
also in some cases retarded, ommatidia surrounding clones
were analysed in the adult eye, after patterning is complete
(Table 2 and Fig. 5). These experiments confirmed the results
that were obtained by looking at rotation in discs. Firstly,
ommatidia around clones are seen to have both chiral forms
and to be able to rotate either clockwise or counterclockwise,
regardless of where they lie with respect to the endogenous
equator of the eye. Secondly, these ommatidia are very often
organised around clearly defined ectopic equators. By
analysing a large number of ptc and pka-C1 clones, we have
found that ectopic equators can apparently run any direction
out of a clone. The amount of non-autonomy (i.e. ectopic
furrow progression) that can be produced by inducing clones
is limited by the size of the zone of competence. Nevertheless,
ectopic equators are commonly seen extending for 5 or more
ommatidial rows with dozens of ommatidia arranged around
4250 D. I. Strutt and M. Mlodzik
Fig. 1. The arrangement of
ommatidial units in the wild-type
eye. Anterior is to the left and dorsal
is up, in this and in all subsequent
figures. (A) An eye disc from a midthird instar stage larva. The position
of the morphogenetic furrow as it
progresses from posterior to anterior
is revealed by expression of a dpplacZ reporter gene (blue). Behind the
furrow, the arrangement of the
assembling ommatidial clusters is
revealed by expression of the
neuronal specific antigen Elav
(brown). The equator is visible as the
axis of symmetry running along the
dorsoventral midline (dotted line).
(B) Confocal image of the equatorial
region of an eye disc, stained for
expression of a svp-lacZ enhancer
trap (red). Expression is initially seen
at highest levels in photoreceptor
neurones R3 and R4, and then later at
lower levels in R1 and R6 (Mlodzik
et al., 1990). In row 4, when
expression is first seen, clusters are
only negligibly rotated from their
original axis of symmetry along the
anteroposterior axis. By row 6,
clusters are rotated 45 degrees
towards the equator (dotted line).
(C) Schematic drawing of an eye disc, illustrating the rotational events that occur during third instar larval life. When ommatidial clusters
emerge from the furrow, their axis of symmetry is in the anteroposterior axis (grey arrows), and they point backwards (if the first photoreceptor
to mature, R8, is defined as initially lying at the apex of the cluster). By row 6, clusters have rotated 45 degrees towards the equator (black
arrows indicate clockwise rotating clusters, red arrows anticlockwise), and maintain this orientation for about 10 rows, before rotating a further
45 degrees. Thus they finally lie 90 degrees from their original axis, pointing towards the equator. During rotation, the ommatidia acquire
asymmetries, leading to the establishment on each side of the equator of opposite chiral forms (i.e. forms that no longer share rotational
symmetry, indicated by tails on arrows). This arrangement is then maintained throughout pupal and adult life. (D) Section of an adult eye (left)
at the R7 level and schematic drawing (right), showing ommatidial arrangement around the equator (yellow line in left panel, green line in
right). Note that ommatidia are all facing towards the equator, and that opposite chiral forms (which do not share rotational symmetry) are
present on each side (represented by black arrows for dorsal identity and red arrows for ventral identity). (E) Magnifications of single R7-level
and R8-level ommatidia, illustrating relationship of arrows used in schematic drawings to actual photoreceptor arrangement. Numbering
indicates identity of individual photoreceptors.
Fig. 2. Homozygous mutant ptc clones induce ectopic
morphogenetic furrows. Third instar eye discs are shown in which
unmarked ptc clones have been induced (see Figs 3-6 for marked
clones). Ommatidial clusters are revealed by staining for the Elav
nuclear antigen (brown), whilst dpp expression (normally in the
furrow) is revealed by blue staining. (A) Disc showing ectopic dpp
expression well anterior of the advancing furrow, and thus outside
the ‘zone of competence’ in which ectopic morphogenetic furrow
progression can occur (see text). (B) Disc showing ectopic dpp
expression and differentiation of ommatidial clusters in the zone of
competence anterior to the advancing endogenous furrow. In an
example such as this, ectopic dpp expression would initially have
been in a ring moving outwards from the clusters of ectopic
photoreceptors, before merging with the advancing furrow. The
behaviour of ptc clones thus observed is identical to the phenotypes
of Hh misexpression and pka-C1 loss-of-function clones (Heberlein
et al., 1995; Pan and Rubin, 1995; Strutt et al., 1995).
Furrow progression and ommatidial polarity 4251
Fig. 3. The direction of furrow progression
determines initial ommatidial polarity. (A) Disc
containing an ectopic furrow produced by induction
of a ptc clone. Photoreceptors in the ommatidial
clusters are stained brown, dpp expression in the
furrow is blue. At the top of the panel, the
endogenous furrow can be seen, progressing
posterior to anterior. This is merged with a curved
ectopic furrow. Emerging photoreceptor clusters
have an axis of symmetry which is coincident with
the direction of furrow progression. (B) Confocal
image of a disc containing a ptc clone just merging
with the endogenous furrow. The clone is marked by
lack of cytoplasmic arm-lacZ staining (red). svplacZ nuclear staining (red) is superimposed with
Elav nuclear staining (green). Ommatidial clusters
which have arisen de novo inside the clone have
random orientation. (C) Section of an adult eye
containing a ptc clone (left) and schematic drawing
(right). Clone is marked by increased levels of
pigment (left) or grey shading (right). Arrows in
schematic drawing are as in Fig. 1, circles indicate
incomplete ommatidia which could not be scored.
Within the clone, randomly oriented ommatidia of
both chiral forms are observed. At the edges of the
clone and outside, greater order is observed, but both
chiral forms are nevertheless present.
Fig. 4. Organisation of ommatidial pattern around a ptc clone in the disc. Upper
panel shows a confocal image of a ptc clone marked by lack of cytoplasmic
arm-lacZ staining (red). svp-lacZ nuclear staining in a subset of photoreceptors
(red) is superimposed with Elav in all photoreceptors (green). The sensitivity of
the nuclear svp-lacZ staining is somewhat reduced by being used in a triple label
with cytoplasmic arm-lacZ and Elav: thus strong svp staining is only seen in
clusters mature enough to have completed the first 45 degree turn (compare Fig.
1B). Lower panel is a schematic drawing, with clone shown by grey shading.
Inset indicates position of clone in disc. Arrows are as in Fig. 1, with their
colour indicating deduced direction of rotation. Circles mark clusters outside the
clone which could not be scored. Within the posterior part of the clone,
ommatidial orientation appears random, but many clusters are incomplete or
have distorted shape, which makes precise scoring difficult. The clone is
sufficiently elongated in the anteroposterior axis, that differentiation in the
anterior part (i.e. furthest from the zone of competence) is occurring as a result
of an anteriorly moving furrow, rather than de novo. A number of clusters on the
dorsal edge are unrotated or only neglibly rotated (grey arrows in lower panel),
compared to more mature neighbours. More dorsally, ventrally and anteriorly,
fields of ommatidia are seen which have rotated in opposite directions. In some
cases, field of oppositely rotated ommatidia are opposed, providing the first
evidence of equator formation (yellow lines in upper, green in lower).
4252 D. I. Strutt and M. Mlodzik
Table 2. Summary of ptc and pka-C1 clones analysed in
sections of adult eyes
ptc clones
Position
No.
No. showing
analysed
No. showing
No. showing
‘hijacking’ of
in detail* ectopic equator(s) multiple equators normal equator
Dorsal
Central
Ventral
Total
13
12
7
32
8
10
3
21
Dorsal
Central
Ventral
Total
13
25
9
47
5
18
4
27
4
7
−
11
n.a.
6
n.a.
6
1
9
1
11
n.a.
7
n.a.
7
pka-C1 clones
*Total number of clones sectioned was >100. Only the ones with good
morphology and angle of section are listed. n.a. = not applicable.
them and, in exceptional cases, large proportions of the eye
field are repatterned.
Interestingly, in the adult, we do not see large numbers of
unrotated ommatidia at the edge of clones, where we can
predict their initial polarity. Instead 90 degree rotation is
seen, as for ommatidia in a wild-type eye. This is in contrast
to our observation in the disc, where clusters are often seen
to be unrotated at the edge of clones. This is consistent with
a view that all ommatidia rotate 90 degrees during their
development, but that the time of rotation is not strictly
fixed.
The position of the equator is not fixed along the
dorsoventral midline
In certain cases, when a clone is induced close to the normal
equator of the disc, clusters anterior to the clone can be
arranged around an ectopic equator which is to one side of the
original equator (Fig. 6A). This can lead to the endogenous
equator being ‘hijacked’ by the ectopic equator, leading to a
single equator positioned away from the dorsoventral midline,
and not necessarily running precisely along the anteroposterior
axis. Thus, the position of the endogenous equator can not be
fixed by a global mechanism.
A similar result can be produced by perturbing the even progression of the endogenous furrow throughout its movement
across the disc, leading to a furrow that has abnormal topology.
Such an effect can be produced by activating the hh signalling
pathway throughout the disc, either by using hypomorphic
viable alleles of ptc (D. S. and M. M., unpublished data), or by
using the tub>y+>hh construct that permits ubiquitous
expression of Hh protein throughout a tissue (Basler and
Struhl, 1994). In such a situation, the furrow does not move
evenly across the disc in the anteroposterior axis. This can lead
to not only the creation of more than one ‘leading edge’ of
furrow progression, but also to the formation of multiple
equators in the disc, around which large fields of ommatidia
are arranged (Fig. 6B).
In summary, these results show that there is not a fixed
equator in the eye disc around which ommatidia organise themselves, but that an equator may occur in any position.
DISCUSSION
Within the highly ordered geometric array of the adult eye,
ommatidia display precise polarity, being arranged in mirrorsymmetric fashion about an equator that runs along the
dorsoventral midline (Dietrich, 1909; Tomlinson and Ready,
1987). Our results provide insights into what mechanisms
underlie the establishment of this polarity.
Direction of furrow progression is a direct
determinant of ommatidial polarity
Our results indicate a central rôle for the furrow as an organiser
of ommatidial polarity. This is reflected in the fact that the
direction of furrow progression determines the initial orientation of each ommatidial cluster. Furthermore, it is likely that
ommatidial units are limited to rotating no more than 90
degrees, as to a first approximation most ommatidia surrounding ptc or pka-C1 clones always show this degree of rotation
(in the adult). Clusters that are not born from a moving furrow
have random orientation, which they seem to maintain
throughout development. This supports the hypothesis that
ommatidia are not able to freely rotate in order to achieve a
polarity that is consistent either with that of their neighbours
or with their position in the disc. Thus the first component of
polarity information that an ommatidium receives by virtue of
being born from a moving furrow is a direct determinant of its
final polarity. We propose therefore that there is no requirement for an anteroposterior positional information system in
the eye imaginal disc to which ommatidia are able to respond
in order to determine their polarity.
How is the direction of ommatidial rotation
determined?
The second component of polarity information that an ommatidium responds to is apparent in the direction that it rotates
and in the chiral form that it subsequently adopts. Normally,
all ommatidia in the dorsal half of the eye will rotate in the
same direction and adopt the same chiral form, whilst all
ommatidia in the ventral half will rotate in the opposite
direction and adopt the opposite chiral form. One possible
explanation for this behaviour would be that ommatidia were
aware of their position in either the dorsal or the ventral half
of the disc. This information could be conveyed by a system
of positional information and the developing clusters would
then be ‘pre-programmed’ to rotate in the appropriate direction
and adopt the correct chiral form. A simpler form of such a
model would be that an ommatidium would merely sense in
which direction the equator (i.e. the dorsoventral midline) lay
and rotate towards it, as has recently been proposed (Ma and
Moses, 1995).
Following induction of ptc and pka-C1 clones, we see
aberrant behaviour of ectopic ommatidial clusters. Firstly, both
within and around clones, we observe ommatidia of both chiral
forms. Secondly, around clones, we observe ommatidia that are
predicted to have rotated in either of the possible directions.
Thirdly, rotation is not always in a consistent direction relative
to the dorsoventral midline for an ommatidium of given
starting polarity. Thus, chiral form and direction of rotation
cannot be directly linked to absolute dorsoventral position in
the disc. Neither can their rotation be correlated with the
direction in which the dorsoventral midline lies. Therefore, it
Furrow progression and ommatidial polarity 4253
can be excluded that the rotational behaviour of these ectopic
ommatidial clusters is a result of their ability to sense their
position in the disc relative to the dorsoventral axis.
Nevertheless, despite the fact that there is no consistent
relationship between direction of rotation and the dorsoventral
axis, ommatidial behaviour is not random. Fields of ommatidia
rotate in the same direction and adopt the same chiral form.
Strikingly these fields are often apposed in such a way as to
give rise to ectopic equators. Furthermore, the ommatidia
arranged around such ectopic equators have the correct chiral
form relative to the arrangement around the endogenous
equator. Such ectopic equators can be oriented in all directions
within the disc, and exhibit identical behaviour whether they
lie in the dorsal or the ventral half of the disc.
The failure of ectopic ommatidia to respect the dorsoventral coordinates of the eye disc could have two possible explanations. The first is that there is no system of dorsoventral
positional information, but instead the cells in the disc epithelium are essentially naive about their position. In this case,
they would gain information about polarity by communication with their neighbours. As the ectopic ommatidia are
isolated from the ‘organised’ ommatidia behind the furrow,
they are unable to adopt a polarity consistent with their
absolute position. The second explanation is that ectopic activation of the hh pathway by induction of ptc or pka-C1 clones
is in some way interfering with or redefining the dorsoventral coordinates of the disc epithelium. This might imply a
central rôle for the hh pathway in defining coordinates on two
axes. Although we cannot definitely exclude either possibility, we favour the first view as being sufficient to account for
our results.
It is interesting to note that quite small clones can permanently shift the position of the equator: if these clones were
causing only a local perturbation in positional values, then it
might be expected that the equator would eventually shift back
to its original axis. The fact that this does not happen, again
supports the hypothesis that dorsoventral positional values are
not globally determined.
as a result of ubiquitous hh pathway activation) then ommatidia
throughout the disc can be organised around multiple equators.
These ‘extra’ equators originate at the leading edges of the
curved furrow (see Fig. 6).
Taking these results together, we conclude that equators are
defined by the leading edge of the furrow and that furrow
topology is the critical factor. With respect to the normal
equator in a wild-type disc, this hypothesis fits well with the
previous experimental observation that ommatidia in the centre
of a row emerge sooner than those further towards the edges
of the disc (Wolff and Ready, 1991). These most mature
ommatidia at the dorsoventral midline could then constitute an
organising centre or node which defines the position of the
equator. This information would then be propagated outwards
to less mature ommatidial clusters (Fig. 7). This model of an
outward sweep of patterning from a node has already been
proposed as the most likely mode of patterning in the eye
imaginal disc (Gubb, 1993). It is a corollary of this model that
ommatidial units can organise themselves, with no input from
external coordinate systems.
The formation of ectopic equators by ptc and pka-C1 clones
can be easily explained by this model. Such clones give rise to
ectopic furrows that are normally curved and also, when they
merge with the endogenous furrow usually produce convex
distortions. This curvature produces leading edges similar to
those that would occur at the posterior edge of the eye disc
when furrow progression first begins (see Fig. 7). The clusters
that emerge from these ectopic leading edges then form nodes
around which ectopic equators are organised. To a large extent,
whether and where a node forms will depend on the shape of
the clone and where it intersects with the normal furrow. Furthermore, it is easy to envisage that a clone could give rise to
multiple nodes which might compete with each other and with
the endogenous equator in attempting to organise adjacent
ommatidia. These factors would account for the lack of regularity with which ectopic equators are formed, and the fact that
not all ommatidia around clones can be clearly seen to be
organised around an equator.
How is the position of an equator determined?
Although we observe equators moving in all directions in the
disc, and sometimes with curved rather than straight paths,
they always begin at the edge of a clone and move outwards.
Therefore, their initial direction of travel corresponds to the
inferred direction of furrow progression. This is consistent
with the observation that ommatidia apparently can only
rotate 90 degrees from their initial axis (which is itself determined by the direction of furrow progression). Thus an
equator must always travel in the same direction as the furrow
itself.
This intimate relationship between the axis of equator
formation and the direction of furrow progression is an
important observation, because it suggests that equator
formation may actually be dependent upon furrow progression.
Two experimental results are particularly informative in this
context. Firstly, the fact that the endogenous equator can be
shifted off its normal course by perturbing furrow progression
(with a small ptc or pka-C1 clone), indicates that there is
nothing special about this equator and that it too is influenced
by furrow progression. Secondly, if the endogenous furrow
does not move evenly throughout development (for instance,
How is polarity information propagated from nodes?
An interesting question is how a node is formed and how information is subsequently propagated outwards. There is clearly
no requirement for a special ‘node-building’ mechanism at the
posterior edge of the disc (where the normal equator begins).
Rather, it is observed that cells anywhere in the eye field are
capable of participating in the interactions that can give rise to
a node (and hence an equator).
Our favoured hypothesis is that when ommatidial clusters
are born from the furrow, they are receptive to signals from
their more mature neighbours which normally would
determine in which direction they should rotate. If their neighbours (lying either posteriorly or laterally) are already
organised relative to an equator, then they also will rotate
appropriately and similarly signal this decision to their neighbours. If no clear signals are received, then a group (or at least
a pair) of adjacent clusters communicate and define an equator
between them. As the clusters already have defined polarity in
one axis (as a consequence of the furrow moving in a defined
direction), this definition of an equator provides them with the
second axis. The clusters then rotate and adopt the correct
chiral form. This group (or pair) of clusters now provides the
4254 D. I. Strutt and M. Mlodzik
node about which other clusters can be recruited into a regular
array. The definition of the node is obviously the critical stage,
but to a large extent this may occur by default, with the most
mature clusters performing this function. Thus equators are
formed by the leading edge of the furrow because this gives
rise to the most mature clusters.
It is interesting to note that a node can also be ‘broken’. This
is the case when a ptc or pka-C1 clone perturbs or stops the
progress of the endogenous equator, as a result of an ectopic
furrow intersecting with the endogenous furrow close to the
dorsoventral midline (Fig. 6A). This again
emphasises the point that the endogenous
equator has no special properties, but itself
is born from a self-organising node.
Further support for the model is provided
by the observation that ommatidial clusters
born from ectopic furrows often seem slow
to rotate, relative to clusters born from the
normal furrow. This delay might well
represent the time needed for ommatidia to
communicate together in order to define a
node. It should also be noted that clusters on
the edges of clones are lying adjacent to
randomly oriented clusters inside the clone,
which may themselves be sending out conflicting signals, a factor that again might
delay rotation. Of course, delays in rotation
around a clone might also represent a
conflict between signals from a global positional information system and a pertubation
Fig. 5. Ectopic equators in the adult eye induced
by ptc clones. Panels on left show sections, the
area of the clone being marked by increased
levels of pigment and ectopic equators being
indicated in yellow. Panels on right are
schematic drawings in which clones are marked
by grey shading and ectopic equators by green
lines; arrows are as in Fig. 1 with colour
indicating chirality, circles mark incomplete
ommatidia which could not be scored. Insets
show position of clone in eye. None of these
sections include the endogenous equator, which
lies ventrally in A, posteriorly in B and dorsally
in C. Note that ommatidia of both chiral forms
are seen in all the sections, and that rotation both
towards and away from the endogenous equator
must have occurred. (A) Clone touching dorsal
edge of eye. An ectopic equator runs out
dorsally, organising five rows of ommatidia.
Ventrally a short equator can also be defined
extending for 2 rows. (B) Large clone lying
centrally just below the dorsoventral midline.
Ectopic equators running largely in the
dorsoventral axis are present both above and
below the clone. Within the section shown, about
5 rows of ommatidia are organised around each
of these equators, but in fact the equators extend
further. The endogenous equator stops at the
posterior edge of the clone (outside section
shown). (C) Ventral clone, giving rise to an
ectopic equator running approximately in the
anteroposterior axis, parallel to the endogenous
equator.
in this system being caused by ectopic activation of the hh
pathway. However, if this were the case, it is surprising how
often ectopic equators are observed (Table 2), rather than
ommatidia adopting an orientation appropriate to their
position in the disc.
At present the molecular nature of the signals involved in
interommatidial communication is unknown. However, there
is increasing evidence that the tissue polarity class of genes
might constitute a signalling system and, as their mutant
phenotypes affect ommatidial polarity, these are excellent can-
Furrow progression and ommatidial polarity 4255
Fig. 6. The position of the
endogenous equator is not
fixed. (A) Upper panel
shows a confocal image of
a ptc clone in the disc,
lower panel shows
schematic drawing.
Staining and labelling is as
for Fig. 4. The endogenous
equator (running from the
posterior edge) is disrupted
when it comes into contact
with clusters born from an
ectopic furrow around the
ptc clone. A single equator
continues anteriorly and
slightly dorsally out of the
clone, around which all of
the clusters anterior to the
clone are organised. Two
other possible small
equators are marked as
extending out of the clone
in the dorsoventral axis,
one of almost intersects
with the endogenous
equator. (B) Disc from a
larva carrying the
tub>y+>hh transgene,
which has been used to
induce ubiquitous Hh expression during first instar larval life. This treatment gives rise to a furrow (marked by dpp expression in blue) which
progresses unevenly in the anteroposterior axis. The ommatidial arrangement is revealed by Elav staining (brown). At least two lines of
symmetry can be seen in the arrangement of the ommatidia, representing two equators (black lines). These equators originate at the leading
edges of the furrow, which we regard as acting as organising centres.
Fig. 7. Model for the formation of the equator. (A) The mature ommatidial clusters at the posterior edge of a wild-type third instar eye disc, as
revealed by Elav staining. The rows of ommatidia are curved, and more clusters are added in successive rows, leading to centre out
development. Arrow indicates position of equator. (B) Schematic drawing, representing the formation of a node at the posterior edge of the
disc. Three consecutive stages of development are shown, from least mature on the left to most mature on the right. Rotational events have
been accelerated for clarity. Initially ommatidial clusters face towards the furrow from which they have just emerged. By row 5-6, the most
mature clusters are rotating away from each other to form an equator, following inter-ommatidial communication. Less mature clusters can then
receive polarity information both by communicating with their neighbours and with more mature clusters lying posteriorly. Note that polarity
information essentially travels from the equator outwards, ensuring that precise packing can be achieved.
4256 D. I. Strutt and M. Mlodzik
didates to be involved (Gubb and García-Bellido, 1982; Vinson
and Adler, 1987; Vinson et al., 1989; Gubb, 1993; Theisen et
al., 1994). A recent analysis of the function of one of this class
of genes (frizzled) in eye development suggests that polarity
information is transmitted outwards from the equator (Zheng
et al., 1995), in a fashion consistent with our view of an
outward sweep of polarity information from a node at the
leading edge of the furrow. An independent analysis of the
effects of ectopic furrows on ommatidial pattern formation also
supports our conclusion that the leading edge of the furrow
serves as an organising centre for equator formation (Chanut
and Heberlein, 1995).
Multiple equators can also be produced in the Drosophila
eye by localised cell death (caused by induction of eyeless
clones) dividing the eye field into two separate regions
(Campos-Ortega, 1980). Although it is not clear what the
mechanism underlying this phenomenon might be, it is nevertheless compatible with the notion that isolated ommatidia are
able to self-organise independently of the global coordinates
of the disc.
CONCLUSIONS
In summary, we propose that ommatidial polarity is defined by
two factors: firstly, the direction of furrow progression and,
secondly, the ability of ommatidia to communicate in a
polarised manner and autonomously organise themselves to
produce an equator. This then defines a node or nucleation
centre about which subsequent pattern is built.
We thank Konrad Basler for flies, Gerry Rubin for providing the
Elav antibody, Steve Cohen and Bill Brook for reading the manuscript, and Ulrike Heberlein, Kevin Moses and Richard Carthew for
sharing unpublished results. D. S. was supported by a Wellcome Trust
Travelling Research Fellowship and an EU Human Capital and
Mobility Fellowship.
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