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Introduction
Genes that contain the homeobox sequence (McGinnis et
al., 1984; Scott and Weiner, 1984) control a range of cell
fates in all eukaryotes examined. They appear to act as tran-
scriptional regulators that are responsible for establishing a
pattern of gene expression appropriate for a given cell type
or developmental region (for recent reviews see Hayashi
and Scott, 1990; Affolter et al., 1990; Andrew and Scott,
1992). In order to understand more fully how homeobox
genes so profoundly affect the fate of a cell during devel-
opment, it will be necessary to identify the main compo-
nents of the pathway downstream of these genes. The search
for downstream genes has been carried out by a variety of
strategies. These include analysing the effect of homeobox
gene mutations on the expression of candidate target genes
(for example, see Hafen et al., 1984; Struhl and White,
1985; Bienz and Tremml, 1988; Winslow et al., 1989;
Reuter et al., 1990) and screening for genetic modifiers of
homeobox gene mutations (Kennison and Russell, 1987;
Kennison and Tamkun, 1988). The approach that we have
taken in searching for genes downstream of rough relies on
enhancer traps, and combines some of the advantages of
both these previous strategies. A similar approach was
recently used to identify potential targets of the Antenna -
pedia gene (Wagner-Bernholz et al., 1991).
The approximately 750 individual ommatidia that form
the compound eye differentiate in a monolayer epithelium
called the eye imaginal disc. Each ommatidium has eight
photoreceptors, and the differentiation of these photore-
ceptors occurs in a precise sequence: first R8 is determined,
then R2 and R5, R3 and R4, R1 and R6, and finally R7. It
is believed that this stereotypical development is regulated
by a series of inductive signals: as each new cell or pair of
cells differentiates, it induces its neighbours to adopt a
specific fate (for reviews see Tomlinson, 1988; Ready,
1989). The product of the rough gene is known to be nec-
essary for the specification of R2 and R5 identity in the
developing imaginal disc (Tomlinson et al., 1988; Basler et
al., 1990; Kimmel et al., 1990). In rough mutants, the pre-
sumptive R2/R5 cells lose some aspects of their normal
identity, and appear to be partially transformed into other
photoreceptor subtypes (Heberlein et al., 1991; Van Vactor
et al., 1991).
In an effort to understand how the activity of rough in
the presumptive cells R2 and R5 leads to their acquiring
that specific fate, we have compared the expression of 101
enhancer trap lines in wild-type and rough−flies. Enhancer
trap lines result from the chromosomal insertion of an E.
coli lacZ gene next to a transcriptional enhancer, thereby
generating a pattern of β-galactosidase expression that
reflects the pattern of expression of the gene at the site of
insertion (O’Kane and Gehring, 1987; see Freeman, 1991,
for recent review). We chose to examine enhancer trap lines
335
Development 116, 335-346 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
In order to identify potential target genes of the rough
homeodomain protein, which is known to specify some
aspects of the R2/R5 photoreceptor subtype in the
Drosophila eye, we have carried out a search for
enhancer trap lines whose expression is rough-depen-
dent. We crossed 101 enhancer traps that are expressed
in the developing eye into a rough mutant background,
and have identified seven lines that have altered
expression patterns. One of these putative rough target
genes is rhomboid, a gene known to be required for
dorsoventral patterning and development of some of the
nervous system in the embryo. We have examined the
role of rhomboid in eye development and find that, while
mutant clones have only a subtle phenotype, ectopic
expression of the gene causes the non-neuronal mystery
cells to be transformed into photoreceptors. We propose
that rhomboid is a part of a partially redundant network
of genes that specify photoreceptor cell fate.
Key words: Drosophila, homeobox, rough, rhomboid, enhancer
trap, retina, photoreceptor.
Summary
Identifying targets of the rough homeobox gene of Drosophila: evidence
that rhomboid functions in eye development
MATTHEW FREEMAN1,*, BRUCE E. KIMMEL2,* and GERALD M. RUBIN
Howard Hughes Medical Institute, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
1Present address: Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
2Present address: Department of Anatomy, University of California, San Francisco, CA 94143, USA
*The first two authors contributed equally to this work
336
that had been previously shown to be expressed early in the
developing photoreceptors, and sceened for lines with
rough-dependent expression in the presumptive R2 and R5
cells. This approach has allowed us to identify several loci
that appear to be downstream of rough in R2 and R5. It is
important to stress that these experiments alone cannot dis-
tinguish between genes that are directly transcriptionally
controlled by rough, and those that are more indirectly
dependent on rough function for their expression.
One of the enhancer traps that we have thus identified as
being downstream of rough is in the previously identified
gene, rhomboid.rhomboid is known to have a function in
several stages of embryogenesis, including the formation of
some ventral structures and parts of the nervous system
(Mayer and Nüsslein-Volhard, 1988; Bier et al., 1990).
Until now there has been no evidence for rhomboid func-
tioning in eye development. We have investigated the role
of rhomboid in photoreceptor differentiation, and our results
suggest that it is part of the process that causes cells to
adopt a photoreceptor fate, although this function appears
to be largely redundant.
Materials and methods
Fly strains and genetics
The enhancer trap lines that were crossed into a rough−back-
ground were isolated in a screen for genes expressed posterior to
the morphogenetic furrow (M. F., U. Gaul, J. S. Heilig, L. S. Hig-
gins, and G. M. R., unpublished data). We followed the proce-
dure described by Mlodzik and Hiromi (1991), but used the plwb
element, which contains the white gene as a dominant marker
(kindly provided by U. Grossniklaus and W. Gehring). The
enhancer trap lines used to examine the identity of the extra pho-
toreceptors were also isolated in this screen. Enhancer trap lines
were crossed into a rox63 background by standard techniques; rox63
is a null allele of rough (Heberlein et al., 1991). Care was taken
to generate fly stocks which were homozygous for individual
enhancer trap lines and rox63, thus eliminating the two-fold dif-
ferences in the level of staining that are observed between het-
erozygous and homozygous individuals. Enhancer traps that were
homozygous lethal were screened as heterozygotes.
rhomboid alleles used in this study include rho7M43 (Mayer and
Nüsslein-Volhard, 1988), kindly provided by C. Nüsslein-Vol-
hard; rhodel1 (Bier et al., 1990), kindly provided by E. Bier; and
the three excision alleles described in the text and shown in Fig.
2A, rhoP∆5,rhoP∆16 andrhoP∆38. These excision alleles were made
by crossing X81 flies to flies carrying a stable source of P trans-
posase (Robertson et al., 1988), and selecting individuals that had
lost the P element, as detected by loss of the white+marker. Lethal
lines thus generated were tested for complementation with
rho7M43, and all new rhomboid alleles were analysed by genomic
DNA blotting (see legend to Fig. 2).
Mitotic clones were induced as described by Tomlinson et al.
(1988). The dominant marker that we used to detect clones of
rhomboid tissue in the adult eye was the P[w]33 element at cyto-
genetic position 70C (our unpublished data). Adult Drosophila
heads were fixed and sectioned as described by Freeman et al.
(1992).
Molecular techniques
All DNA manipulation and analysis were done according to the
protocols of Sambrook et al. (1989). The sevenless-rhomboid
ectopic expression construct was made in several steps. First, a
rhomboid fragment was amplified using a two-step PCR protocol
(see Higuchi, 1989, for a description of this procedure). The
primers were designed so that a novel XbaI site was added 16
nucleotides 5′of the initiating AUG codon, and the rhomboid frag-
ment extended as far as the HindIII site 3′of the transcription
unit, as shown in Fig. 2A. The rhomboid coding region was
sequenced in order to ensure that no PCR errors were incorpo-
rated into the ectopic expression constructs. This XbaI to HindIII
rhomboid cassette was then cloned downstream of a fragment con-
taining the sevenless promoter (−967 bp to +89 bp, Bowtell et al.,
1988), giving a transcriptional fusion. This promoter/transcript
fragment was then cloned into a plasmid that contained sevenless
enhancer sequences (+90 bp to +9.3 kb; see Bowtell et al., 1989)
inserted into pDM30, a P-element transformation vector (Mismer
and Rubin, 1987). We also made a variant of this construct, in
which most of the 3′untranslated region of the rhomboid gene
had been deleted. We found no difference between transformants
with the two constructs, and have treated them as equivalent when
producing flies with multiple sev-rho copies.
The heat shock-rhomboid construct was made by cloning the
XbaI to HindIII rhomboid cassette described above downstream
of a 306 nucleotide fragment of the hsp70 gene (corresponding to
coordinates 189 to −495 in Fig. 4 of Ingolia et al., 1980), which
includes the heat-shock element and the TATA box. This HS-rho
fusion was then cloned into the transformation vector pw8 (Kle-
menz et al., 1987). P-element transformation (Spradling and
Rubin, 1982) with both sev-rho and HS-rho was done by standard
procedures. Several transformants were isolated for each con-
struct; flies with multiple copies of sev-rho were made by recom-
bining different insertions onto the same chromosome, and by
crossing insertions on different chromosomes into the same fly.
Activity and immunostaining of imaginal discs and embyros
The expression of enhancer traps in imaginal discs and embryos
was assayed by an X-gal (5-bromo-4-chloro-3-indolyl β-D-galac-
topyranoside) activity stain for β-galactosidase (Bellen et al.,
1989; Wagner-Bernholz et al., 1991). Eye imaginal discs were
immunostained as described previously (Freeman et al., 1992),
except for the disc shown in Fig. 3D which was fixed in 4%
paraformaldehyde in the presence of 1% NP-40; all subsequent
stages were done by standard methods. Embyros were fixed and
immunostained as described by Patel et al. (1987). The anti-neu-
roglian is a monoclonal antibody (Hortsch et al., 1990), and the
supernatant was used at a dilution of 1:5. mAbro1 is a monoclonal
antibody raised against the rough protein (Kimmel et al., 1990),
and was used at a dilution of 1:10,000. Anti-β-galactosidase was
bought from Promega, and was used at 1:500; Biorad horserad-
ish peroxidase-coupled goat anti-mouse IgG was used at 1:300.
Embryonic cuticle preparations
Cuticle preparations were done according to the protocol of
Wieschaus and Nüsslein-Volhard (1986). Embryos were removed
from their vitelline membranes prior to fixation and were mounted
in 1:1 Hoyer’s mountant:lactic acid.
Producing an antibody against rhomboid
Using standard PCR techniques, a 458 bp DNA fragment encod-
ing the N-terminal 153 amino acids of the rhomboid protein was
generated and ligated into the pATH11 trpE fusion protein vector
(Koerner et al., 1991). The resulting plasmid produced a trpE-
rhomboid fusion protein of approximately 60×103Mrwhich was
used as an immunogen for monoclonal antibody production by
standard methods (Harlow and Lane, 1988). Two anti-rhomboid
monoclonal antibodies were isolated and called mAbrho1 and
mAbrho2.
M. Freeman, B. E. Kimmel and G. M. Rubin
337Rhomboid and eye development
Results
Using enhancer traps to search for genes downstream of
rough
We have looked for genes that might lie downstream of
rough by examining the effect of its absence on the
expression pattern of a collection of enhancer traps. The
rough gene encodes a homeodomain protein that controls
some of those aspects of R2 and R5 development that pro-
vide them with an identity different from the other six pho-
toreceptor cells. Rough protein is expressed in cells R2, R5,
R3 and R4, but the rough gene is only required in cells R2
and R5 for normal eye development (Tomlinson et al.,
1988; Kimmel et al., 1990). When comparing the
expression of the enhancer traps in wild-type and rough
larvae, we screened for the most obvious differences: for
example, greatly reduced expression in R2 and R5. More
subtle reductions or enhancements of expression may have
been missed.
As listed in Table 1, only seven of the 101 enhancer traps
tested have a significantly altered expression pattern in the
absence of rough function. Of these, four (A135, AE33, P7,
X81) are lines that are normally expressed in R2 and R5,
and are therefore candidates for rough-dependent genes that
might play a part in the specification of the R2/R5 subtype.
The other three lines (A65, O8, S1) show alterations in cells
other than R2 and R5, and these are most easily explained
as reflecting aspects of the rough phenotype. Interestingly,
three of the lines that might be downstream of rough in
cells R2 and R5 (AE33, P7, X81) are normally expressed
predominantly in photoreceptor precursors R8, R2 and R5:
expression is first seen in cell R8 near the morphogenetic
furrow, and cells R2 and R5 begin to express the marker a
few rows more posteriorly - at about the position where
they start to express neural antigens. All three of these
enhancer traps have reduced β-galactosidase expression in
R2 and R5 in the rough mutant eye disc. The best exam-
ples of this are AE33 and X81, whose expression patterns
are shown in Fig. 1. In total, 3 of the 8 enhancer trap lines
that are expressed in cells R8, R2 and R5, are affected by
loss of rough.
Effects in cells other than R2 and R5
The effects of removing wild-type rough function in cells
other than R2 and R5 are likely to reflect indirect conse-
quences of the loss of rough, due to the abnormal devel-
opment of rough−discs. Recently, it has been demonstrated
(Heberlein et al., 1991; Van Vactor et al., 1991) that addi-
tional cells expressing R8-specific markers differentiate in
many of the ommatidia of an eye disc missing rough func-
tion, and this effect can be seen with some of the enhancer
traps that we have examined. For example, in rough−discs
Fig. 1. The effect of rough on enhancer trap expression. Groups of
ommatidia from third instar eye imaginal discs are shown, stained
for the expression of the β-galactosidase reporter gene by X-gal
activity stain. (A and C) The enhancer trap line X81 is dependent
on rough function in cells R2 and R5: compare the expression of
the reporter gene in wild-type (A) and rough−(C) discs. Instead of
the three cell groups (R8, R2, R5) stained in wild-type discs, only
one, or occasionally two cells, per cluster are stained in the rough−
disc. The arrow in C points to an example of a pair of R8-like
cells staining (see text for explanation). Although this
phenomenon is seen in 30-40% of ommatidia, it is less frequent
towards the dorsal and ventral edges of the disc than closer to the
equator. The panels show regions away from the equator - hence
the apparent rarity of the R8 pairs. Staining in the presumptive
cells R2 and R5 is abolished. The X81 enhancer trap also directs
β-galactosidase expression in photoreceptors in the pupal retina.
As in the larval disc, R2, R5 and R8 stain most strongly in 40 hour
X81 pupal retinas stained with X-gal (data not shown). (B and D)
The enhancer trap line AE33 is also normally expressed in cells
R8, R2 and R5 (B), and in a rough−disc shows the same effect as
X81: staining is abolished in cells R2 and R5, but remains in R8
(D).
Table 1. Seven enhancer trap lines whose expression pattern is altered in the absence of rough function
Enhancer trap # Chromosome position rough+expression pattern rough expression pattern
A65 32B1-2 R8 strong; R1-R7 weak 25% of ommatidia have 2 strongly staining cells.
A135 40A1-4 R1-R8; Fades at disc posterior Reduced expression and fades sooner.
AE33 51C5-6 R8, R2 and R5 R2 and R5 expression reduced.
O8 32F1-2 R3, R4, R7 and cone cells R3 and R4 expression altered
P7 90D4-5 R8, R2, R5 strong; R1, R3, R4, R6, R7 weak R2 and R5 expression reduced.
S1 42F1-2 R3, R4 strong; R1, R2, R5-R8 weak Early R3 and R4 expression missing?
X81 62A1-2 R8, R2, R5 strong; R1, R3, R4, R6, R7 weak R2 and R5 expression is reduced or absent.
338
expressing the R8/R2/R5-specific enhancer trap lines AE33,
P7, and X81, it is quite clear that the β-galactosidase
expression in the R2 and R5 precursor cells is reduced. In
these lines a single R8 cell is seen near the furrow, but fur-
ther posterior in the disc, approximately 40% of the omma-
tidial clusters have two cell nuclei which stain strongly (Fig.
1C). Furthermore, the line A65 normally expresses β-galac-
tosidase most strongly in the R8 precursor cell and more
weakly in the other cells, while in a rough disc many dou-
blets of cell nuclei staining with the intensity of the R8 pre-
cursor cell are observed.
All of the enhancer traps that express β-galactosidase in
the R3 and R4 photoreceptor reflect the developmental
defects that occur in the R3 and R4 photoreceptors in a
rough−eye disc. However, the O8 and S1 enhancer traps
show additional defects with respect to the development of
the R3 and R4 photoreceptors. Thus in S1, the early β-
galactosidase expression in R3 and R4 appears to be com-
pletely absent in a rough background. In a rough−disc,
the O8 enhancer trap directs expression of β-galactosidase
in pairs of nuclei near the morphogenetic furrow which
resemble R3 and R4 precursors; further posterior only one
of these nuclei remains apical, and the other appears to sink
basally. These basal nuclei have an odd cylindrical mor-
phology which is never observed for wild-type β-galac-
tosidase-expressing nuclei, and they may represent cells that
are undergoing cell death. On rare occasions, O8 is also
expressed in more than two cells per ommatidium in a
rough disc.
The X81 enhancer trap line is an insertion into the
rhomboid locus
The X81 enhancer trap insertion was localised by in situ
hybridisation to cytological position 62A1,2; this is the
same location as the previously characterised gene, rhom -
boid, which functions in embryonic development (Bier et
al., 1990). We isolated genomic DNA flanking the X81
insertion point, and mapped the P element insertion to
between 100 and 150 nucleotides 5′of the predicted tran-
scriptional start site of rhomboid (see Fig. 2A). Flies
homozygous for the X81 insertion are wild-type.
The embryonic pattern of β-galactosidase expression in
the enhancer trap line X81 (Fig. 2B-2D) is similar in almost
all detail to the rhomboid RNA expression described by
Bier et al. (1990). The only difference is that we have seen
no evidence of a longitudinal dorsal stripe of expression in
the blastoderm. Outside the embryo, a major difference in
the expression pattern of X81 and the rholac1 enhancer trap
studied by Bier et al. (1990) is that X81 is expressed in the
eye imaginal disc, as described above, while rholac1 is not.
(Another enhancer trap in rhomboid is also expressed in the
eye disc; U. Gaul and G. M. R., unpublished observations.)
A possible reason for this difference is that, although the
X81 and rholac1 elements are only about 100 nucleotides
apart, they are in different transcriptional orientations. The
two enhancer traps might therefore detect significantly dif-
ferent components of the complete rhomboid expression
pattern.
Loss of function rhomboid mutations
Upon mobilisation, P-elements frequently excise impre-
cisely, causing the formation of small deletions of genomic
DNA around their insertion point (Daniels et al., 1985). We
took advantage of this phenomenon to generate new rhom -
boid mutations. We screened 897 lines of flies from which
the white gene marker carried by the P-element had been
lost, and from these we isolated 40 lethal lines, all of which
failed to complement the rho7M43 mutation (Mayer and
Nüsslein-Volhard, 1988). These 40 new rhomboid alleles
were then screened by genomic DNA blotting to identify
deletions that extended as far as the translational initiation
site. Two such excision lines were found, rhoP∆5and
rhoP∆38; a third line, rhoP∆16, is also very likely to remove
the translational start site (see legend to Fig. 2). Since these
alleles are deletions that extend from at or near the point
of insertion of the X81 enhancer trap element, through the
whole 5′end of the rhomboid transcript and into the coding
region of the gene, they almost certainly represent complete
loss-of-function mutations of rhomboid. Previously exist-
ing rhomboid mutations have not been sufficiently charac-
terised to know whether they result in a complete loss of
function. Embryonic cuticle preparations of each of these
new rhomboid mutations look identical, and they appear
not to be significantly different from those described for the
rho7M43 mutation (Mayer and Nüsslein-Volhard, 1988; see
Fig. 2E and 2F).
rhomboid appears to act downstream of rough in the
developing eye; however, we have been unable to detect
any evidence of a genetic interaction between mutations in
these two genes. In particular, a decrease in the gene dosage
of rhomboid has no apparent effect on the phenotype of a
weak rough allele (data not shown).
The distribution of the rhomboid protein
We have raised monoclonal antibodies against a fusion pro-
tein containing the N-terminal 153 amino acids of the rhom -
boid protein and used these to investigate the expression of
the rhomboid protein (Fig. 3). Although the staining that
we observe is rather weak, the rhomboid protein expression
that we observed in embryos (data not shown) corresponds
closely to rhomboid RNA expression, as described by Bier
et al. (1990).
The sequence of the rhomboid protein indicates that it
has several membrane spanning domains, and this leads to
the prediction that it is a membrane-associated protein (Bier
et al., 1990). Our data show that the rhomboid protein in
embryos appears to be predominantly localised in vesicles
(Fig. 3A). The nature of these vesicles is not known, but
they resemble those in which the Drosophila sevenless,
boss,scabrous and wingless proteins are found (Tomlinson
et al., 1987; Krämer et al., 1991; Baker et al., 1990; Van
den Heuvel et al., 1989). Sevenless is a transmembrane
receptor protein, and boss is its membrane-associated
ligand; these vesicles in the R7 precursor cell have been
shown to contain internalized ligand/receptor complexes
(Krämer et al., 1991). Scabrous and wingless are both
secreted proteins.
Rhomboid protein is also detected in the third instar eye
imaginal disc, where it is limited to the developing omma-
tidia (Fig. 3B). This confirms that the X81 enhancer trap
expression does reflect real expression of rhomboid in the
M. Freeman, B. E. Kimmel and G. M. Rubin
339Rhomboid and eye development
developing eye disc. Under normal fixation conditions, the
staining is punctate, and is reminiscent of the vesicle stain-
ing in the embryo, making it difficult to distinguish pre-
cisely in which cells the rhomboid protein is expressed (Fig.
3C). However, it appears to be limited to only a subset of
the developing cells and, in discs fixed in the presence of
the detergent NP-40 (see Materials and Methods), this
subset can be identified as cells 8, 2 and 5 (Fig. 3D). This
observation is consistent with the expression of the X81
enhancer trap, which is transcribed in predominantly cells
8, 2 and 5.
Mitotic clones of rhomboid in the adult eye
In order to investigate the role of rhomboid in eye devel-
opment, we have characterised the phenotype of rhomboid
mutations in the eye. Since all rhomboid mutations are
lethal, this can only be done by generating mitotic clones
of homozygous rhomboid tissue in an otherwise het-
erozygous fly. An eye color marker was used to allow the
identification of the homozygous mutant clones in the eye.
We have made mitotic clones with both of our null rhom -
boid mutations (rhoP∆5and rhoP∆38), and with two exist-
ing mutations, rho7M43 and rhodel1, and find them all to have
Fig. 2. (A) Map of the region around rhomboid. The X81 enhancer trap element is inserted between 100 and 150 nucleotides 5′of the
presumed transcriptional start site of the rhomboid gene (Bier et al., 1990), and they are in the same transcriptional orientation.
Sequencing of the genomic DNA encompassing the gene indicated that there is one more small intron in the coding sequence than has
been previously reported. This intron is between nucleotides 1114 and 1115 in the coordinates used by Bier et al. (1990), and is
approximately 140 nucleotides long; it contains an EcoRI site. The extent of small deletions caused by the imprecise excision of the
enhancer trap element is shown: the hatched bars represent sequences that are missing in the mutations indicated, and the open bars
indicate the uncertainty in mapping the ends of the deletions. rhoP∆16 leaves the left end of the element intact, and deletes to between an
XmnI site 17 nucleotides 5′of the presumed initiating ATG codon, and an SphI site 88 nucleotides 3′of the ATG; it is therefore likely, but
not certain, that rhoP∆16 deletes the beginning of the protein. rhoP∆5and rhoP∆38 both delete the whole P element, and may delete DNA as
far as the EcoRI site to the left of the insert; they both delete as far to the right as between the SphI site 88 nucleotides 3′of the ATG, and
an EcoRV site 267 nucleotides 3′of the ATG: both these mutations therefore remove the N terminus of the protein, as well as the entire 5′
end of the transcript. (B, BamHI; H, HindIII; R, EcoRI.). (B-D) X-gal staining to indicate the expression pattern of the X81 enhancer trap
in the embryo (anterior to the left). (B - ventral view). In the gastrulating embryo, the expression is limited to a few rows of cells on either
side of the ventral furrow; soon after the stage shown here, the expression narrows to a single row of cells at the ventral midline. There is
also expression in a broad band in the head region. (C - lateral view) At the germband extended stage, the mesectoderm is stained. (D -
ventral view) In a germband retracted embryo staining is seen in ventral epidermal stripes in each segment, and in a subset of cells in the
midline of the nervous system (an example is indicated by the arrow). Not seen in this view are the dorsal stripes, which are similar to the
ventral ones. (E) An embryonic cuticle preparation of a wild-type embryo, and (F) of a rhoP∆5homozygous embryo (anterior to the left).
Note the characteristic fusions of the denticle belts in the rhomboid embryo (indicated by an arrow), as well as the abnormal head
skeleton.
340
an identical, subtle phenotype in the adult eye. There is a
very slight disruption of the ommatidial array associated
with the clones (Fig. 4). This defect most often occurs at
the boundary between wild-type and mutant tissue (as
shown in Fig. 4). The significance of this boundary effect
is not clear, nor is the underlying cause of the phenotype.
It is worth stressing that, while this phenotype is very subtle
and only affects a small number of ommatidia, it is highly
reproducible and is likely to represent the true loss-of-func-
tion phenotype of rhomboid: four different mutations,
induced in different genetic backgrounds, show the same
effect, and two of those mutations are almost certainly com-
plete protein nulls.
Ectopic expression of rhomboid
In order to examine further what role rhomboid might have
in ommatidial determination, we tested the effect of alter-
ing its expression pattern in the developing eye. We chose
two different ectopic expression procedures (Fig. 5A). In
the first experiment we linked the rhomboid coding region
to the enhancer and promoter of the sevenless gene and
introduced them into flies by P-element transformation.
These control sequences have been previously shown to
confer the sevenless expression pattern on heterologous
genes (Basler et al., 1989; Bowtell et al., 1989; Kimmel et
al., 1990). sevenless is expressed at least transiently in all
the cells of the developing ommatidium except for R8, R2
and R5, so transformants that are wild type for their own
rhomboid gene, and which contain a sevenless-rhomboid
(sev-rho) fusion gene, will express rhomboid in all omma-
tidial cells including the cone cells and mystery cells (Fig.
5B). The second ectopic expression construct that we made
was to put the rhomboid coding sequences under the con-
trol of the hsp70 promoter. Upon heat shock, transformants
carrying this construct will express rhomboid in all cells,
including the uncommitted cells surrounding each omma-
tidium.
Ectopic expression under the sevenless promoter
sev-rho transformants have rough eyes. The degree of
roughness appears to be quite sensitive to the dose of the
rhomboid gene since different transformant lines show
varying amounts of disorder, and all transformants show
increasing roughness with increasing copy number of the
sev-rho construct. In general two copies are enough to show
clear roughness, and when six copies are combined in the
same fly there are severe disruptions in the pigment lattice
and lens structures. Sections through transformant eyes
show that the primary defect in sev-rho eyes is the pres-
ence of one, two and occasionally three extra photorecep-
tor cells (compare Fig. 5C and 5D). These extra cells have
the characteristic morphology of the outer photoreceptors
R1-R6. Often the addition of these cells to the ommatid-
ium does not greatly disrupt the overall architecture, and in
these cases the additional cells can be seen to be in the
vicinity of cells R3 and R4. The penetrance of this extra
cell phenotype depends on the dose of the sev-rho con-
struct: Fig. 5D shows a section through an eye containing
two copies, in which about 30% of the ommatidia have
extra cells; up to four copies produces a similar phenotype,
although the proportion of ommatidia with extra cells
increases to about 70%; when the copy number is over four,
other defects are frequent. These include missing pigment
cells leading to fusions between adjacent ommatidia, loss
M. Freeman, B. E. Kimmel and G. M. Rubin
Fig. 4. The phenotype of
rhomboid mutations in the eye.
Mitotic clones of several different
rhomboid mutations were
sectioned, and all showed the
same subtle but reproducible
phenotype. Typically, a small
number of ommatidia were
missing, leading to a slight local
roughening in the clone. Often a
whole row of ommatidia is
missing in the clone, and this is
shown in this 2 micron section: the
edge of a clone is pictured, with
the mutant, white , tissue to the
left of the panel, and the wild-type,
white+, tissue towards the right. At
the clone boundary a row of
ommatidia from the wild-type
tissue terminates. We have tried to
indicate this phenomenon more
clearly by drawing a line through
each ommatidial row.
341Rhomboid and eye development
Fig. 5. Ectopic expression of rhomboid. (A) The two misexpression constructs. The sev-rho construct includes a sevenless enhancer fragment
(speckled), and a sevenless promoter fragment (hatched), linked to the rhomboid transcription unit from which the first, non-coding, exon and
the first intron have been removed (see Materials and Methods). The HS-rho construct has a fragment containing the sequences necessary to
confer heatshock inducibility upon a heterologous gene (see Materials and Methods) linked to the same rhomboid fragment as the sev-rho
construct. (B) The sev-rho misexpression experiment. rhomboid appears to be predominantly expressed in cells R8, R2 and R5; sevenless is
expressed in all the other cells of the developing ommatidia (Tomlinson et al., 1987); in sev-rho flies, which are wild type for their own copy
of the rhomboid gene, the gene is expressed in all the cells. These diagrams represent all the cells that are associated with the ommatidia
throughout larval development, rather than any particular stage of their differentiation. (C) A 2 micron tangential section through a wild-type
adult eye. Note that in this plane of section seven photoreceptors can be seen in each ommatidium; the photoreceptors are easily identified by
their rhabdomeres, the dark organelles used for light trapping. The identity of each photoreceptor can be determined by its position in the
asymmetric trapezoid, and these identities are indicated. (D) A similar section through a fly carrying two copies of the sev-rho construct: note
that several of the ommatidia have an extra outer photoreceptor (examples of such ommatidia are indicated with arrows). (E) A section
through a HS-rho eye, which was heatshocked as a third instar larva. One of the ommatidia (indicated by an arrow) has an extra outer
photoreceptor that is indistinguishable from those in sev-rho eyes. (F-H) Third instar eye imaginal discs stained with an antibody against
neuroglian, which is a ubiquitous neural antigen expressed early in neuronal differentiation (Hortsch et al., 1990); in each case the
morphogenetic furrow is to the left. (F) A wild-type disc. Cells R8, R2, and R5 are indicated. Cells R3 and R4, adjacent to R2 and R5, can be
seen to stain weakly at this stage in some of the clusters. (G) A sev-rho disc from a larva with two copies of the sev-rho construct. Cells in the
position of the mystery cells that are expressing neuroglian are indicated with arrowheads. (H) A disc from a larva with six copies of the sev-
rho construct. In these discs, a greater proportion of ommatidia have extra cells differentiating neuronally, and more overall disruption of the
disc is apparent. The broad arrowheads indicate some examples of neuronally differentiating cells in the mystery cell position; the arrow
indicates an extra cell, between clusters, that is undergoing neural differentiation.
342
of regularity of spacing and orientation, and occasional
extra photoreceptors with inner photoreceptor morphology;
the lens defects apparent in eyes from flies with six copies
of sev-rho suggest that there are also disruptions in cone
cell development when the ectopic dose of rhomboid
becomes very high.
In order to understand what the initial defects were in
the development of eyes of sev-rho flies, we examined the
earliest stages of their development in the eye imaginal disc.
During the third larval instar, a dorso-ventral indentation,
known as the morphogenetic furrow, sweeps anteriorly
across this monolayer epithelium. As cells emerge from the
posterior of the furrow, they begin to show morphological
signs of differentiating, and they begin to express neural
antigens. Cells are recruited into the developing ommatidia
in a precise sequence, and every differentiating cell can be
recognised as it develops (Tomlinson, 1988; Ready, 1989).
In discs containing two copies of the sev-rho construct,
extra cells undergoing neural differentiation can be seen in
many of the clusters. This is most easily observed at the
stage when three cells (R8, R2 and R5), and then, soon
after, five cells (R8, R2, R5, R3 and R4), are normally
expressing neural antigens. The additional one or two cells
are in the position of the mystery cells in wild-type discs,
adjacent to cells R3 and R4 (Fig. 5G). The mystery cells
are so called since their ultimate fate is unclear. In wild-
type discs they join the precluster of the first five photore-
ceptor precursors, but they never express neural antigens,
and eventually they leave the cluster and appear to rejoin
the pool of uncommitted cells (Tomlinson et al., 1987). In
sev-rho flies, some of the mystery cells never leave the clus-
ter, and differentiate as morphologically normal outer pho-
toreceptor cells. The proportion of ommatidia with these
transformed mystery cells correlates well with that seen in
sections of adult eyes. With two copies of sev-rho, the trans-
formation of the mystery cells is the only defect that we
can detect in sev-rho discs. With higher copy numbers,
other defects become apparent including abnormal spacing
and orientation, and the occasional additional photorecep-
tor that is not in the position of a mystery cell (Fig. 5H),
which may represent a different cell adopting an inappro-
priate photoreceptor fate.
Ectopic expression under the hsp70 promoter
Flies carrying the HS-rho construct are wild type at 25°C.
However, upon a series of heat shocks during the third
M. Freeman, B. E. Kimmel and G. M. Rubin
Fig. 6. The identity of the extra photoreceptors in sev-rho imaginal discs. A to C are wild-type discs carrying enhancer trap insertions
specific for particular cells; E to G are discs with three copies of the sev-rho construct carrying the same enhancer trap lines. All these
discs were immunostained for β-galactosidase, and the morphogenetic furrow is to the left. A and E show discs carrying an enhancer trap
in the seven-up gene (Mlodzik et al., 1990). This marker is expressed only in cells R3, R4, R1 and R6, as is shown in A; E in sev-rho
discs, many ommatidia have additional staining cells adjacent to cells R3 and R4 (see arrows). B and F show discs carrying an enhancer
trap called O32 (our unpublished data). This is expressed predominantly in cells R3, R4 and R7, but in the region near the furrow, where
this field is from, only cells R3 and R4 are stained (B); (F) in sev-rho discs additional cells adjacent to R3 and R4 are seen to express O32
in many ommatidia (example indicated by an arrow). C and G show discs carrying the AE33 enhancer trap (described in this work),
which is predominantly expressed in cells R8, R2, and R5 (C); sev-rho discs do not have additional AE33-expressing cells (G). D and H
are wild-type and sev-rho discs, respectively, stained with mAbro1, an antibody against the rough protein. D shows the normal expression
pattern in cells R2, R5, R3 and R4. H shows the staining in sev-rho discs: many ommatidia are found to have extra rough-expressing
cells, adjacent to cells R3 and R4, in the mystery cell position (indicated by arrows).
343Rhomboid and eye development
larval instar, when photoreceptor determination is occur-
ring, a stripe of slight roughness is induced in the adult eye.
Sections through this rough portion of the eye indicate an
ommatidial phenotype that is indistinguishable from that of
sev-rho flies: one or two extra outer photoreceptors are
found in the vicinity of R3 and R4 (Fig. 5E). We presume
that these are also transformed mystery cells. In order to
see this effect, quite severe heat shock regimes are required,
for example four 30 minute pulses over a period of 6.5
hours to larvae carrying two copies of the HS-rho construct.
Even then, the penetrance of this extra cell phenotype is
low: typically two or three abnormal ommatidia in an eye.
We do not know why the HS-rho constructs appear less
effective at producing the transformed mystery cell pheno-
type than the sev-rho constructs, but it may be that the level
per cell of ectopic rhomboid expression is quite low in HS-
rho discs. Interestingly, even though rhomboid is ectopi-
cally expressed in all cells, including all the uncommitted
cells in the disc, we see no evidence for any cells other
than mystery cells being transformed into photoreceptors.
The identity of the transformed mystery cells
We have used cell-type-specific markers to try and deter-
mine the identity of the transformed mystery cells in sev-
rho flies. These markers are enhancer trap lines that have
previously been shown to express the E. coli lacZ gene in
various subsets of the developing photoreceptors (M. F., U.
Gaul, J. S. Heilig, L. S. Higgins, G. M. R., unpublished;
Mlodzik et al., 1990). By crossing these enhancer traps into
flies carrying sev-rho, we were able to examine which
markers are expressed by the extra cells, and therefore to
determine their identity. The transformed mystery cells
express an enhancer trap in the seven-up gene, which is
specific to R3, R4, R1 and R6 (Fig. 6E) and one that is
specific to cells R3, R4 and R7 (Fig. 6F); they do not
express an enhancer trap specific to cells R8, R2 and R5
(Fig. 6G). Furthermore, using an antibody against the rough
protein, we find that the transformed cells express rough.
Normally, only four cells per ommatidium (R2, R5, R3, R4)
express rough protein posterior to the morphogenetic
furrow (Kimmel et al., 1990). However, in sev-rho discs,
we often see five or six cells per ommatidium staining, and
the additional cells are in the mystery cell position (Fig.
6H). On the basis of these results, it appears as if the trans-
formed mystery cells have an identity closest to cells R3
and R4.
Discussion
The identification of homeobox target genes should eluci-
date some of the mechanisms by which a cell chooses
between alternative fates. The approach that we have used
to identify genes downstream of the rough gene is to cross
enhancer traps, which were previously known to be
expressed in the developing ommatidium, into a rough
mutant background and thus to look for genes whose
expression was rough-dependent. rough has previously
been shown to be required only in cells R2 and R5, and it
has a critical role in the specification of their fate (Tom-
linson et al., 1988; Basler et al., 1990; Kimmel et al., 1990;
Heberlein et al., 1991). Our strategy made no assumptions
about what genes might be regulated by rough, and allowed
us to examine directly whether the transcription of a gene
was rough-dependent. Our search has identified several can-
didates, and we have characterised further the role of one
of these, rhomboid, during eye development. We have
shown that rhomboid is expressed in the developing omma-
tidia, where its expression in cells R2 and R5 is rough-
dependent; rhomboid mutations subtly affect normal eye
development, and its ectopic expression causes a profound
fate change in the mystery cells, which are transformed into
photoreceptors.
Cell identities and rough target genes
It is important to note that cells R2 and R5 do not differ-
entiate normally in rough mutants. Nevertheless, in most
cases the two cells that would become R2 and R5 in a wild-
type disc can still be identified by their position in the
ommatidial precluster, and they still differentiate as neu-
rons (Tomlinson et al., 1988). In rough mutants these two
presumptive R2/R5 cells lose at least part of their normal
identity, and instead appear to acquire a variety of abnor-
mal fates. For example, Heberlein et al. (1991) have shown
that many of these cells take on some characteristics of R3,
R4, R1 and R6 cells in that they express and become depen-
dent on the product of the seven-up gene, which is normally
required and expressed in only cells R3, R4, R1 and R6.
In other cases, one of the presumptive R2/R5 cells acquires
an R8-like fate. Van Vactor et al. (1991) observed ectopic
boss expression in many of the presumptive R2/R5 cells,
suggesting a more frequent transformation toward R8. One
interpretation of these results is that in the absence of rough
the presumptive R2/R5 cells are receptive to inappropriate
cues, and their fate may then be decided stochastically or
by small variations in local environment. This view is con-
sistent with the variability in the composition of individual
ommatidia seen in the eyes of rough adults. It is also pos-
sible that the presumptive R2/R5 cells adopt a hybrid cell
fate, simultaneously expressing cell-type-specific markers
that are never co-expressed in the same cell in wild type.
Additional evidence that rough controls some part of the
R2/R5 fate comes from the results of its ectopic expression
under the sevenless promoter: this causes the presumptive
R7 cell to be transformed into an outer photoreceptor
(Basler et al., 1990; Kimmel et al., 1990), although the
specific subtype of this transformed cell is unclear. Our data
suggest that rough does not control all aspects of the R2/R5
identity. In our screen, 5 out of 8 lines, which are normally
expressed in the three cells R8, R2 and R5, were unaffected
by the loss of rough function.
In total only 4 out of 101 lines that we examined
appeared to detect genes that were rough targets in cells
R2 and R5. We have found that approximately 5% of all
enhancer trap lines are expressed at moderate to high levels
in the developing ommatidia (M. F., U. Gaul, J. S. Heilig,
L. S. Higgins, G. M. R., unpublished). Taken together, these
numbers suggest that the total number of genes dependent
on rough for R2 and R5 specification is not large. Previ-
ous analysis of the rough phenotype indicates that, mini-
mally, rough must regulate the genes necessary for pro-
viding the inductive signal to specify the photoreceptor
344
identity of cells R3 and R4, and for specifying some part
of the outer photoreceptor subtype identity.
In the simplest view, it would be expected that those
genes that were required for the R2/R5 subtype, and which
were downstream of rough, would be expressed in only
cells R2 and R5: although rough is expressed in cells R2,
R5, R3 and R4, it is not required in R3 and R4 (Tomlin-
son et al., 1988), and furthermore, those genes that specify
the R2/R5 identity seem unlikely to be expressed in R3/R4.
However, in the enhancer trap screen on which this study
was based, no lines that were expressed only in R2 and R5
were isolated. Instead, most of those lines that do appear
to be downstream of rough in R2 and R5 are expressed in
the triplet of cells R8, R2 and R5. This could indicate a
relatively close developmental relationship between R8 and
R2/R5, a conclusion previously suggested by a characteri-
sation of the Star gene in eye development (Heberlein and
Rubin, 1991): Star is required in cells R8, R2 and R5 for
normal ommatidial differentiation. Genes identified by the
enhancer trap lines that are expressed in R8, R2 and R5
must be regulated by factors other than rough in R8, since
rough is not significantly expressed in that cell.
The role of rhomboid in eye development
The X81 enhancer trap, whose expression is dependent on
rough, is an insertion at the rhomboid locus. This was the
first indication that rhomboid, known to be necessary for
embryonic development (Mayer and Nüsslein-Volhard,
1988; Bier et al., 1990), was also likely to have a role in
eye development. We have confirmed that the enhancer trap
expression pattern does reflect real expression of rhomboid
in the eye imaginal disc. Although our antibody does not
stain strongly enough to characterise unambiguously the
details of the protein’s expression - either because the
amount of rhomboid protein is low, or because the anti-
body is of low affinity - it is quite clear that rhomboid is
expressed in the developing photoreceptors, beginning early
in their differentiation. It is likely that the expression is lim-
ited to cells R8, R2 and R5, as detected by the enhancer
trap.
When misexpressed under the sevenless promoter/
enhancer, rhomboid profoundly alters the fate of the mys-
tery cells, causing them to become outer photoreceptors,
with characteristics of cells R3 and R4. This result suggests
that rhomboid may function in the process by which at least
some cells acquire a photoreceptor fate. When ectopically
expressed under the control of the heat shock promoter, and
therefore expressed in all cells, it is still only the mystery
cells that inappropriately adopt a photoreceptor fate. Even
in wild-type discs, the mystery cells undergo some of the
early stages of photoreceptor differentiation, although they
ultimately appear to rejoin the pool of uncommitted cells.
They undergo similar morphological changes to the pho-
toreceptors of the early five-cell precluster; they express
sevenless; and they are particularly susceptible to acquiring
a photoreceptor fate in a variety of mutants (Tomlinson et
al., 1987; Mlodzik et al., 1990; Fischer-Vize et al., 1992;
Freeman et al., 1992). Thus the mystery cells seem to be
developmentally quite close to photoreceptors in the
precluster and ectopic expression of rhomboid is sufficient
to alter their fate.
Although there is a dramatic effect of ectopically
expressing rhomboid, loss of rhomboid function has a rel-
atively mild effect in the eye. The only visible phenotype
in mutant clones is a slight disruption of the ommatidial
array. The easiest way to reconcile this observation with
the ectopic expression results is to suggest that rhomboid
is part of a largely redundant network of gene products:
because of this redundancy, the removal of rhomboid only
has minor effects.
There are two alternative explanations for our ectopic
expression results: in one rhomboid acts autonomously, and
its expression in the mystery cell under the sevenless pro-
moter causes the cell’s transformation into a photoreceptor;
in the other view, rhomboid acts non-autonomously, in
which case its ectopic expression in cells R3 and R4 induces
the adjacent mystery cells to adopt a photoreceptor fate. It
is tempting to speculate that, since rhomboid appears to be
downstream of rough in cells R2 and R5, it may be part of
the inductive signal presented to R3 and R4, and this would
favour the non-autonomous model of rhomboid action. In
this view, rhomboid is part of a complex network of over-
lapping signals that are used to specify a photoreceptor fate
in the developing eye. In sev-rho flies, cells R3 and R4
ectopically express rhomboid, thus presenting an inappro-
priate inductive signal to their neighbours, the mystery
cells, causing the transformation of the latter into R3/R4
like cells. Since rhomboid appears to be only part of a
mechanism that causes photoreceptor determination, its
ectopic expression only affects the mystery cells, which
may already express many of the gene products necessary
for this developmental pathway, and therefore only need a
slight ‘push’ to adopt that fate. This model provides a
testable hypothesis: the question of whether or not rhom -
boid acts autonomously can be addressed by mosaic analy-
sis. Unfortunately, the sev-rho transformation does not pro-
duce a penetrant-enough phenotype to allow a simple
analysis in the adult eye, so this experiment will have to
be done by analysing clones in the developing eye disc -
which will be technically challenging.
Functional redundancy of rhomboid
rhomboid encodes a protein whose function appears to be
partially redundant in the eye. While it may not be possi-
ble to select for fully-redundant functions in evolution,
largely overlapping functions may be quite common. This
could account for the observation that a large majority of
mutations in genes adjacent to enhancer traps expressed in
the developing eye, have no obvious effect on eye devel-
opment (our unpublished observations). The view that a
majority of genes will produce clear phenotypes that illu-
minate their normal function may be heavily biased by the
fact that most genes characterised to date have been ini-
tially identified by having a clear mutant phenotype. As
more genes are isolated by reverse-genetic techniques,
which do not rely on a phenotype, a more accurate picture
of the extent of redundancy of developmental mechanisms
should emerge. It is notable that in those cases where small
regions of the genome have been extensively analysed for
transcription units and genes with detectable phenotypes,
there appears to be a two- to three-fold excess of transcripts
over phenotypes (for example see Hall et al., 1983). There
M. Freeman, B. E. Kimmel and G. M. Rubin
345Rhomboid and eye development
are some attractive theoretical features of a system with
such overlapping functions. These include robustness, since
a small perturbation is less likely to affect a process dele-
teriously; and the ability to adapt relatively easily, there
being less selection pressure against a change in any single
gene in such a redundant network.
We are grateful to Dave Hackett for injecting P-element trans-
formation constructs and Todd Laverty for chromosome in situ
hybridisations. We thank Mellissa Cobb, Iswar Hariharan, Bruce
Hay and Ulrike Heberlein for their useful comments on the man-
uscript.
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347Rhomboid and eye development
Fig. 3. The expression of rhomboid protein in the embryo and the eye imaginal disc. Immunohistochemical staining with the monoclonal
antibody mAbrho1 is shown. (A) A view of two lateral stripes of rhomboid expression in a germband retracted embryo; note that
rhomboid protein is largely restricted to vesicles (arrows). (B) rhomboid protein is expressed in the third instar eye imaginal disc,
posterior to the morphogenetic furrow (anterior to the left, arrow marks position of furrow). C and D are higher magnification views of a
portion of an eye disc. (C) Under normal fixation conditions, rhomboid staining in the eye disc is punctate, making it difficult to
determine the identity of individual cells. (D) Using a fix that includes the detergent NP-40 (see Materials and Methods), the rhomboid
protein is apparently somewhat solubilised, allowing a clearer identification of the cells in which it is expressed; an example of the
characteristic triplet of cells R8, R2 and R5 is indicated.
dev9082 fig. 3 colour tipin
