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The spitz gene is required for photoreceptor determination in the Drosophila eye where it interacts with the EGF receptor
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Mechanisms of Development48 (1994) 25-33
The spitz gene is required for photoreceptor determination in the Drosophila eye where it interacts with the EGF receptor Matthew Freeman* MRC Laboratory of Molecular Biology. Hills Road. Cambridge CB2 2QH. UK
Received 22 April 1994; accepted 24 May 1994
Abstract
Little is known about the mechanisms by which photoreceptors other than R7 are determined during Drosophila eye development. By looking for mutations that modify the phenotype caused by ectopic expression of the rhomboid gene in the eye, I have discovered that the spitz gene is required for photoreceptor determination. Mosaic analysis suggests that spitz, which encodes a TGFc~ homoiogue, produces a diffusible signal during ommatidial development. Other members of the spitz group and the EGF receptor also interact with sev-rho, in a pattern that suggests a model in which rhomboid can act as a mediator of a ligand-receptor interaction between spitz and Egfr in the developing eye. These data suggest that photoreceptors other than R7 use a Ras i signalling pathway activated by the spitz/Egfr interaction, in a manner analagous to the Rasl pathway activated by boss/sevenless in photoreceptor R7. Keywords: Drosophila; EGF receptor; Eye development; rhomboid; Signal transduction; spitz
1. Introduction
The individual facets or ommatidia of the Drosophila compound eye each contain eight photoreceptors, comprising three distinct classes based on their morphology and which opsin they express. Photoreceptors R I - R 6 express the Rhl opsin and have large rhabdomeres (the light gathering organelles); R7 expresses Rh3 or Rh4 and has a small rhabdomere; and R8 also has a small rhabdomere and expresses an unknown opsin. The photoreceptors are determined during the third larval instar in the eye imaginal disc. A morphogenetic furrow sweeps anteriorly across the disc leaving the developing ommatidia in its wake. The photoreceptors in each ommatidium develop in a strict sequence, starting with R8 and finishing with R7, and there is evidence that they are recruited by a series of inductive interactions (see Tomlinson, 1988; Ready, 1989; Banerjee and Zipursky, 1990, for reviews on eye development). The main components of the signalling pathway by which R7 is recruited have recently been identified and they c o m *Tel.: 0223 402234; Fax: 0223 412142.
prise a Rasl dependent signal transduction cascade, initiated by the interaction between the sevenless receptor tyrosine kinase and its ligand, boss (see review by Kr~imer and Cagan, 1994). Much less is known about the molecular mechanisms that are responsible for the determination of the other photoreceptors. However, clonal analysis has shown that although the sevenless tyrosine kinase is not required by any cell other than R7 (Harris et al., 1976; Campos-Ortega et al., 1979; Tomlinson and Ready, 1987), the Rasl protein is essential for the formation of all photoreceptors (Simon et al., 1991). This suggests that a Rasl dependent signalling pathway may be central to all photoreceptor determination but that it is activated by some receptor other than sevenless in the non-R7 photoreceptors. A candidate receptor is the Drosophila epidermal growth factor receptor (Livneh et al., 1985; Wadsworth et al., 1985), also a tyrosine kinase receptor that can activate the Rasl pathway (Simon, et ai., 1991). The EGF receptor is pleiotropic (Clifford and Schiipbach, 1989; Clifford and Schiipbach, 1992; Raz and Shilo, 1992) and is needed for cell proliferation in the eye imaginal disc (Xu and Rubin, 1993), making
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M. Freeman./Mech. Dev. 48 (1994) 25-33
clonal analysis difficult. However, it does have later functions in eye development: dominant Ellipse alleles of the EGF receptor gene (Egfr) disrupt the number of ommatidia that form (Baker and Rubin, 1989; Baker and Rubin, 1992), and analysis of very small clones indicates that the EGF receptor is probably required in all photoreceptors (Xu and Rubin, 1993). The genes of the spitz group, spitz, rhomboid, Star and pointed, appear to act in a common pathway at several stages in development (Mayer and Nusslein-Volhard, 1988). Recent evidence suggests that this pathway includes the EGF receptor and the spitz group may act 'to mediate EGF receptor signalling. For example, Egfr is required for the establishment of ventral ectoderm as are the members of the spitz group and genetic interactions suggest that they comprise a common pathway (Raz and Shilo, 1993). Similar interactions have been observed in the oocyte and the wing (Ruohola-Baker et al., 1993; Sturtevant et al., 1993). The specific functions of the spitz group genes are not all clear, spitz itself encodes a homolog of the vertebrate EGF receptor ligand, TGFa (Rutledge et al., 1992) and is probably the ligand for the EGF receptor in the ventrolateral signalling pathway, although it is unknown if it acts in a membrane-tethered or a released form. This view is supported by recent biochemical evidence that spitz protein can bind to and activate the EGF receptor in tissue culture cells (R. Schweitzer and B. Shilo, personal communication). The product of the rhomboid gene is predicted to have multiple transmembrane domains (Bier et al., 1990), but is not similar to any known protein (although a similar gene has recently been identified in the nematode by the C. elegans sequencing project - - EMBL accession number Z29117). It is striking that rhomboid is expressed specifically in those cells which are missing in spitz group mutants (Bier et al., 1990), whereas the products of Egfr and spitz are both more widely expressed (Zak et al., 1990; Rutledge et al., 1992; Zak and Shilo, 1992). These observations led to the view that rhomboid may provide spatial cues to the proposed spitz/Egfr interaction and there is recent evidence supporting this model in the oocyte and the wing (Ruohola-Baker et al., 1993; Sturtevant et al., 1993), although the mechanism by which rhomboid might facilitate the interaction is unknown. Star also encodes a transmembrane protein of unknown function (Kolodkin et al., 1994) and pointed encodes two related proteins with ETS domains which are presumed to be transcription factors (Kl/imbt, 1993). In this paper, I examine the role of spitz, the EGF receptor, and other members of the spitz group in the developing eye. Previous work has shown that rhomboid is expressed in a subset of developing photoreceptors and clones of rhomboid mutant tissue show subtle defects in the eye (Freeman et al., 1992a). These defects are most easily interpreted as the occasional failure of om-
matidia to be established in the absence of rhomboid, but the low penetrance of this phenotype has made it difficult to understand the role of rhomboid in eye development. When misexpressed under the promoter of the eye-specific gene sevenless, rhomboid has profound effects: the mystery cells are transformed into photoreceptors and other cells are also affected (Freeman et al., 1992a). These sev-rho flies have a rough eye and I have looked for mutations that modify this phenotype. Mutations in spitz, Egfr, Rasl and another gene in the Rasl pathway, Sos, affect the sev-rho phenotype, supporting the notion that rhomboid can act in an Egfr pathway with the spitz group in the developing eye. Since spitz itself has not previously been shown to act in photoreceptors, I have investigated its role in their development and find that it is necessary for the determination of all photoreceptors. Clonal analysis indicates that it is probably acts as a diffusible factor of rather limited range. I propose that the interaction between spitz and the EGF receptor activates the Rasl dependent pathway necessary for photoreceptor determination in cells other than R7. 2. Results
2.1. Loss of function spitz mutations suppress the sevrhomboid phenotype The eye defects caused by sev-rho (Fig. 1) are dose sensitive; that is, they become more extreme with increasing copy number of the sev-rhomboid transposon (data not shown). This implies that the severity of the defects is sensitive to the degree of signalling mediated by the ectopic rhomboid, which in turn suggests that a 50% reduction in dose of other members of the signalling pathway might significantly alter the phenotype. I have tested many mutations for the ability to suppress or enhance the sev-rho rough eye (Table 1) and those which appear to be involved in the pathway are described below. spitz mutations strongly suppress the sev-rho phenotype in a haplo-insufficient manner (Fig. 1). spitz is a recessive mutation: that is, flies which have only one wild-type copy of the gene are indistinguishable from wild-type flies. In contrast, one wild-type copy of spitz is not sufficient to support normal levels of rhomboid signalling in sev-rho eyes: three loss-of-function alleles dominantly suppressed the sev-rho rough eye, spi r25 and spi AI4 strongly, and spi °Ege more weakly. Sections through such eyes indicate that all the defects caused by sev-rho are equivalently suppressed. This result suggests that in order for sev-rho to change the fates of the mystery cells into photoreceptors and to cause the other defects, spitz gene product is required in nearly wildtype doses: halving the dose of spitz significantly reduces the degree of transformation.
M. Freeman./Mech. Dev. 48 (1994) 25-33
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Table 1 Mutations tested for genetic interaction with sev-rhomboid Genotype
Interaction with sev-rho
spiAU/+ spiTeJ/+ spiOege/+ SetS/+
S5671/+ EgfrEt/+ EgfrE3/+
0 (additive) 0 (additive)
EgfdI/+
Egfrf2/+ Egfrf4/ +
Fig. !. Mutations in the spitz gene dominantly suppress the sevrhomboid phenotype. A and C show a scanning electron micrograph and a 2 t~m section, respectively, through an eye of a fly carrying three copies of the sev-rhomboid transposon (sev-rho3 flies). The rough eye is caused primarily by the presence of supernumerary photoreceptor cells but also by missing pigment cells and misorientations of the ommatidia. In C, the photoreceptors are identified by their round, dark organelles, called rhabdomeres, which consist of the light trapping membranes; the pigment cells are identified by the refractive pigment granules within them and by the characteristic lattice which they form around the ommatidia. Arrowheads indicate ommatidia with extra photoreceptors. B and D show similar views of a fly of the genotype spiXt4/+;sev-rho3/+, that is one with three copies of the sev-rho transposon and with only 50% of the dose of the wild-type spitz gene product (spitz At4 appears to be close to a null mutation). The reduction in spitz dose has returned the eye to a near wild-type phenotype, with a regular array of ommatidia, the stereotypical trapezoid arrangement of the seven photoreceptors visible in this plane and the unbroken lattice of pigment cells. The rescue is not always complete, but is always unambiguous. In panel D, a slightly misorientated ommatidium is indicated by an arrowhead.
2.2. spitz is required f o r photoreceptor determination spitz is a n e m b r y o n i c l e t h a l m u t a t i o n , s o in o r d e r t o d i s c o v e r w h e t h e r it a c t s in w i l d - t y p e eye d e v e l o p m e n t , I h a v e m a d e c l o n e s o f spi-/spi- t i s s u e in t h e eye b y m i t o t i c r e c o m b i n a t i o n . T h e s e spitz- c l o n e s s h o w severely d i s r u p t e d p h o t o r e c e p t o r d e v e l o p m e n t , t h e m a i n d e fects b e i n g t h e loss o f p h o t o r e c e p t o r s a n d t h e loss o f
Egfrfa/+ Egfrf7/ + Egfrft 3/+ Egfrf24/+ EgfrtOptot/+
-/0
pnte/+ RasletB/+ Rasle2F/+ Sose°O/+
0
-/0
Sose2H/+ Sose4a/+
-/0
),an i/+ yanel+ Gap1 st6/+ Gap1 s:/+ drkSu(Sev)Stt/+
++ +++ 0 0 0
rafm%
o
faf o4/+ fafl ~Os/+ argosgilS/+ argosgilt5/+
0 0 0 0
groFP2/+ simSS/+ simHg/+
0 0 0
rafltU7/+ rlt/+ rltOa/+
0 0 0
Standard cross procedures were used to produce flies that were heterozygous for the mutations shown and which carried two or three copies of the sev-rho transposon (see Experimental procedures). Symbols indicating the results are as follows: 0, no detectable interaction; - - - , strong suppression of sev-rho phenotype; - - , moderate suppression; - , weak suppression; + + +, strong enhancement of sevrho phenotype, + +, moderate enhancement. The nomenclature of the alleles used is as described in Lindsley and Zimm (1992), where possible, and otherwise follows the convention of earlier publications. w h o l e o m m a t i d i a (Fig. 2). spitz- p h o t o r e c e p t o r s a r e f o u n d , i m p l y i n g t h a t n o t all p h o t o r e c e p t o r s n e e d t o m a k e s p i t z in o r d e r t o d e v e l o p n o r m a l l y . S i n c e o m m a t i d i a a t t h e b o r d e r b e t w e e n w i l d - t y p e a n d spitz- tissue c o n t a i n a m o s a i c o f spitz + a n d spitz- cells, it is
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M. Freeman./Mech. Dev. 48 (1994) 25-33 Table 2 Mosaic analysis of the spitz "414mutation in the eye
Fig. 2. Mitotic clones of spitz- tissue have reduced numbers of photoreeeptors. A white- spitz- clone within a w + spi ÷ background is shown; the clone is seen as the tissue lacking the refractive pigment granules. In the spitz- region, many ommatidia have fewer than normal photoreceptors (examples indicated by arrowheads) and there are a reduced number of total ommatidia as well, compared to the surrounding wild-type tissue. An example of a mosaic, normally constructed ommatidium, of the kind analysed in the mosaic analysis (see Table 2), is labelled to show that cells R3 and R5 are w ÷ (identified by the presence of black pigment granules adjacent to the rhabdomere), while cells RI, R2, R4, R6 and R7 are w-. R8 resides in the basal region of the retina and cannot be scored in this relatively apical section.
p o s s i b l e t o a s k w h i c h cells a r e r e q u i r e d t o b e spitz + t o make a phenotypically wild-type ommatidium. Such a m o s a i c a n a l y s i s s h o w s t h a t t h e o n l y cell t h a t m u s t b e spitz + is t h e R 8 cell, w h i c h is t h e first p h o t o r e c e p t o r t o b e d e t e r m i n e d ( T a b l e 2). T h e r e is a l s o a c l e a r f u n c t i o n in t h e n e x t t w o cells t o b e d e t e r m i n e d , R 2 a n d R 5 , t h o u g h t h e r e q u i r e m e n t is n o t a b s o l u t e . T h e n e x t t w o cells, R 3 a n d R 4 , a l s o a p p e a r t o s h o w a s l i g h t r e q u i r e m e n t t o e x p r e s s spitz a n d t h e l a s t t h r e e cells, R 1, R 6 a n d R7, show no requirement.
2.3. The E G F receptor and genes in its signal transduction pathway interact genetically with spitz and sev-rho in the eye S i n c e s p i t z is p r o b a b l y a l i g a n d f o r t h e E G F r e c e p t o r in t h e e m b r y o a n d t h e E G F r e c e p t o r is r e q u i r e d f o r eye d e v e l o p m e n t , I t e s t e d w h e t h e r Eg)Cr i n t e r a c t s in t h e eye
Photoreceptor
,%w- spi(n = 153)
%w- spi + (n = 86)
RI R2 R3 R4 R5 R6 R7 R8
50 I1 36 20 10 54 47 0.7
44 31 43 34 28 40 42 0
Mosaic analysis was performed in a standard way (for example see Tomlinson et al. 1988), except that clones were induced using the FLP/FRT system instead of by X-ray (see Experimental procedures). Phenotypically wild-type ommatidia that contained both w + and wphotoreceptors were scored and the individual genotype of all eight photoreceptors was noted (see Fig. 2 for example). Two experiments were carried out, the first with spitz AI4 clones, and the second, a control, with wild-type clones. In the first experiment (middle column), 153 phenotypically wild-type ommatidia in spitz- clones were analysed. All but one had w + (and therefore spi +) R8 photoreceptors, indicating that there is essentially an absolute requirement for R8 to be wild-type in order to form a normal ommatidium. The one case where a w- (and therefore spi-) R8 cell was found could be due to a small amount of leakiness in the spi AI4 mutation. This is an EMS allele and has not been characterised. Although it behaves genetically like a null, a Small amount of residual function cannot be ruled out. The w- R8 cell could also indicate that there is a very small probability of producing a normal ommatidium in the absence of spitz in R8, possibly by diffusion of sufficient spitz protein from neighbouring ommatidia (see discussion); or the w- R8 could be an artefact caused by a missing section. Not withstanding this case, it is reasonable to conclude that, in general, R8 needs to be wild-type. It is clear that there are significantly fewer than expected mutant cells R2 and R5 (compare the spi- clone with the control wild-type clone), indicating that there is a partial requirement for spitz in these cells. There is also a suggestion of a slight requirement in cells R3 and R4 (the next pair to become determined) but it is not clear whether the observed differences are significant in this case. In the wild-type control experiment, 86 ommatidia with w ÷ R8 cells were scored to determine whether a minor degree of lineage relationship between R8 and R2/R5 is sufficient to account for the reduced number ofspi- R2 and R5 cells observed in the spitz- clones. As can be seen in the third column, there is a detectable lineage relationship between R2/R5 and R8, indicated by the reduced number of wR2/R5 cells compared to other photoreceptors, when R8 is w + but this reduction is much less than that seen in the spitz experiment. This confirms that the observed requirement for spitz in R2 and R5 is a real effect, not a consequence of the relatedness of these cells with R8.
w i t h spitz, s e v - r h o a n d o t h e r m e m b e r s o f t h e spitz group. I f spitz e n c o d e s a l i g a n d f o r t h e E G F r e c e p t o r in t h e eye, a g e n e t i c i n t e r a c t i o n b e t w e e n t h e m w o u l d b e exp e c t e d . R e d u c i n g t h e level o f b o t h spitz a n d Egfr b y h a l f (ie. flies o f t h e g e n o t y p e spi +/+ Egfr) p r o d u c e d n o d e t e c t a b l e p h e n o t y p e . H o w e v e r , spitz w a s f o u n d t o b e a d o m i n a n t s u p p r e s s o r o f Ellipse alleles o f Egfr (Fig. 3). Although the suppression does not have a dramatic
M. Freeman./Mech. Dev. 48 (1994) 25-33
Fig. 3. Interactions with Ellipse. Scanning electron micrographs of from flies of the following genotypes: A. Eg]'rea!/+. B. spiAU+/+Egfr EBI. The Ellipse B! allele of Egfr causes a dominant rough eye phenotype, A, which is characterised by disorganised ommatidia and a reduction in size of the eye. B. Reduction of the dose of spitz by 50%, by making the fly heterozygous for a spitz loss-offunction mutation causes some suppression of the Ellipse phenotype: this is seen as a significant increase in the overall size of the eye but is most obvious when ommatidia are counted (Table 3).
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eyes
effect on the morphology of the eye, it is clear when the number of ommatidia are counted (Table 3). Reduction by half of the level of spitz increases the mean number of ommatidia in Elp/+ eyes from 432 to 630 (wild-type eyes have approximately 750 ommatidia). The exact nature of Ellipse mutations is not clear but they are dominant mutations in the EGF receptor and are believed to cause hyperactive signalling by the receptor in its normal cells, rather than ectopic signalling (Baker and Rubin, 1989; Baker and Rubin, 1992; Zak and Shilo, 1992). The individual ommatidia are mainly wild type in Elp eyes, but their number is dramatically reduced. The observation that reducing the amount of spitz protein suppresses the phenotype of these mutations is consistent with spitz being a ligand for the EGF receptor. The model that ectopic rhomboid activates an EGF receptor signalling pathway is supported by the observation that loss-of-function mutations in Egfr dominantly suppress the sev-rho phenotype (Fig. 4B), indicating
Table 3 Spit: suppresses the EIp phenotype Genotype
No. of ommatidia (mean ± S.D.)
EgfreS//+ spiAt4+/+Egfr es!
432 ± 18 (n = 6) 630 + 54 (n = 6)
The total number of ommatidia in eyes of the indicated genotypes were counted. In each case the control flies were grown in parallel to the experimental flies to minimise any variability in growth conditions that might effect the phenotype, spi AI4 behaves genetically as a null allele. The differences are significant at p < 0.001 by two-tailed t-test.
Fig. 4. Further interactions with sev-rhomboid. Eyes of the following genotypes are shown. A. sev-rho3/+. B. Egfr/~/+; sev-rho3/+. C. Rasletb/sev-rho3. D. Sose°d/+;sev-rho3/+. E. S2tS/+;sev-rho3/+. A. As seen in Fig. IA, three copies of the sev-rho transposon causes a rough eye. B. Reduction in the dose of the EGF receptor by 50% significantly reduces the severity of the sev-rho phenotype. Similarly, reduction in the doses of the Rasl gene (C) and the Sos gene (D) also suppress the sev-rho phenotype. E. Reduction in the dose of the Star gene is a very effective suppressor of the sev-rho phenotype: the eyes of these flies are almost completely wild-type. The dominant rough-eye phenotype of Star itself is extremely weak (see Fig. 5A) and is hardly detectable in these flies.
that near-normal levels of Egfr are required for ectopic rhomboid to produce its effects. A range of Egfr- alleles was tested and in most cases the degree of suppression of sev-rho correlated with the severity of the allele (see Table 1). Many of the genes in the Egfr signal transduction pathway have been identified (Simon et al., 1991; Dickson et al., 1992; Gaul et al., 1992; Simon et al., 1993; Brunner et al., 1994) and I have looked for genetic interactions between sev-rho and several of them. Of these genes, loss-of-function mutations in the Rasl gene and the Sos gene also dominantly suppressed the sev-rho phenotype (Fig 4C and D), while no interaction was detected between sev-rho and Gap1, drk, Rafl or rolled. This indicates that Rasl and Sos probably are in the same pathway as rhomboid, as predicted if rhomboid participates in EGF receptor signalling. In contrast to these results, Sturtevant et al. (1993) a found that Gap1 was an effective enhancer of ectopically expressed rhomboid in the wing but that mutations in Sos did not affect the wing phenotype. It should be noted that the lack of an interaction with members of the EGF receptor signalling pathway does not indicate that these components are not involved: this kind of dominant interaction only identifies genes whose products are close enough to
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M. Freeman./Mech. Dev. 48 (1994) 25-33
Fig. 5. Interactions with Star. Eyes of the following genotypes are shown. A and E. $218/+. B and F. S218/spiAI4. C and G. S218/Egfr f2. D and H. S218/EgfrEnt. The top row of panels show scanning electron micrographs and the bottom row, 2 #m sections through adult eyes. A and E. Star mutations have a weak eye phenotype that varies between undetectable and barely rough; in sections an occasional missing photoreceptor is seen as illustrated by the arrowhead in E. B and F. The Star phenotype is enhanced by a 50% reduction in dose of the spitz gene. F shows that this enhancement takes the form of a higher proportion of ommatidia with missing photoreceptors. C and G. A reduction of the EGF receptor dose also enhances the Star phenotype. D and H. There is a strongly synergistic interaction between Star and Ellipse mutations of the EGF receptor. The eyes of these flies are severely reduced in size and extremely rough. This phenotype is much more severe than a simple addition of the dominant effects of Elp and Star (see Figs 3A and 5A), indicating an interaction between these two mutations. Sections show that there are a greatly reduced number of photoreceptors in all ommatidia.
being rate limiting that a 50% reduction in gene dose reduces the overall signalling level significantly.
2.4. Star acts in the same pathway Star is a n o t h e r m e m b e r o f the spitz g r o u p that interacts with sev-rho in the eye. Star d o m i n a n t l y suppressed all aspects o f the sev-rho p h e n o t y p e very effectively (Fig. 4E). Again, a 50% reduction in Star gene dose leads to a greatly reduced sev-rho phenotype. I have also examined genetic interactions in the eye between Star, spitz a n d Egfr (Fig. 5). H e t e r o z y g o u s Star adults have very slightly rough eyes, but when the dose o f Egfr or spitz is reduced by 50%, the rough eye is enhanced. There is also a synergistic interaction between Star and Ellipse alleles o f Egfr: both give d o m i n a n t rough eyes, but the d o u b l e heterozygote shows a p h e n o t y p e m o r e severe than w o u l d be expected from a simple additive effect. These S +/+ Egfr e eyes are very
rough and reduced in size and sections t h r o u g h them show that most o m m a t i d i a have a reduced n u m b e r o f p h o t o r e c e p t o r s (Fig 5D a n d H). There are also defects in orientation, spacing, a n d pigment cell a n d bristle numbers. It is n o t clear why b o t h loss-of-function a n d gain-of function m u t a t i o n s in Egfr should enhance the Star p h e n o t y p e b u t the degree o f e n h a n c e m e n t is so different that different mechanisms m a y be involved in each case. Nevertheless, the o b s e r v a t i o n that Star shows these genetic interactions with spitz and Egfr further supports the view that these molecules share a p a t h w a y in the eye.
2.5. yan mutations enhance sev-rhomboid The only interaction observed between sev-rho a n d a gene other than Egfr or a m e m b e r o f the spitz g r o u p was with yan, a negative r e g u l a t o r o f p h o t o r e c e p t o r determ i n a t i o n that encodes a potential transcription factor
M. Freeman./Mech. Dev. 48 (1994) 25-33
Fig. 6. Mutations in yah enhance the sev-rho phenotype. The figure shows a scanning electron micrograph, A, and a 2 /zm section, B, through eyes from flies of the genotypeyanP/+;sev-rho3/+. Comparing this phenotypewith that of sev-rho3/+(Figs 1A and 4A), it is clear that there is a considerable increase in the severity of the sev-rho phenotype when the dose of yan is reduced. The section shows that now almost all ommatidiahave 2 or 3 extra photoreceptors.There are also frequent missing pigment cells and ommatidial misorientations, though these defects could be secondary to the extra-photoreceptor phenotype.
with an ETS domain (Lai and Rubin, 1992). Heterozygous yah mutations severely enhance the sevrho phenotype (Fig. 6). Sections through sev-rho/yan eyes show that the extra roughness is caused by an enhancement of all the defects seen in sev-rho/+ eyes. Most ommatidia have between two and four extra photoreceptors, there are many missing pigment cells and no trace of normal orientation or spacing is found. These observations are consistent with the view that yan may act as a repressor of photoreceptor development: when its dose is reduced, the additional photoreceptor recruitment that occurs in sev-rho eyes is greatly increased. 3. Discussion
3.1. The function of spitz in the eye The data presented here show that spitz is necessary for wild-type photoreceptor development. The mosaic analysis indicates that the only cell that must make spitz protein is the first to be determined, R8, and there is a progressively decreasing requirement in the subsequent photoreceptors to produce it. A model arises from this data, coupled with the predicted protein sequence of spitz (Rutledge et al., 1992), which suggests that it may function as a diffusible factor, and the observed requirement for the EGF receptor during photoreceptor determination (Xu and Rubin, 1993). The spitz protein may act as a diffusible ligand which triggers an EGFreceptor-dependent pathway necessary for photorecep-
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tors to be determined. The observed partial requirement of spitz to be produced by cells R2 and R5 is not easily explained by a model in which a precise interaction occurs between membrane bound ligand and its receptor, but might be expected if the ligand is diffusible. As long as a presumptive photoreceptor is presented with sufficient spitz protein, the cell which produces the signal is not critical; what is important is the local concentration. There would be an absolute requirement for spitz protein to be produced by the first cell to start developing (R8), since without that, the next cells (R2 and R5) could not receive any signal, but after that point there is no requirement for any particular cell to be spitz + as long as there are enough cells to keep the overall level high enough. The partial requirement in cells R2 and R5 suggests that the spitz produced by R8 is not sufficient and as R2 and R5 are also early to develop and, like R8, have a central location in the ommatidium, they are the prime additional source of spitz ligand. It is significant that no normal ommatidia were found in which only R8 was wild-type and that there were a reduced number of ommatidia where only R8 plus one other cell was wildtype (compared to control wild-type clones; data not shown). This suggests that two to three wild-type cells are required to produce a sufficient local concentration of spitz protein for all photoreceptors to develop normally. The mosaic analysis also allows a limit to be set on the range that the spitz signal can diffuse. Since there is essentially an absolute requirement for spitz to be expressed in R8 to make a normal ommatidium, sufficient spitz must not be able to diffuse in from neighbouring wild-type tissue. Therefore spitz protein appears to be able to move between cells within an ommatidium (up to about two cell diameters) but not between ommatidia, which would require a diffusion range of four to five cell diameters. 3.2. Mediation of the Egfr pathway by rhomboid The investigation of rhomboid during oogenesis has led to the conclusion that it encodes a cell autonomous molecule in the membrane that mediates the interaction between the EGF receptor in the dorsal follicle cells and its ligand produced by the oocyte (Ruohola-Baker et al., 1993). There is now convincing evidence that the ligand in that case is the product of the gurken gene which, like spitz, is a molecule similar to TGFc~ (NeumanSilberberg and Schiipbach, 1993). Another study has looked at the role of rhomboid in the developing wing, where it regulates the formation of wing vein(Sturtevant et al., 1993). In that case, rhomboid protein appears to mediate an interaction between the EGF receptor and the spitz protein. Similarly, the ectopic expression of rhomboid in the developing eye activates a spitz/Egfr signalling pathway, providing further evidence that rhomboid acts in this pathway. In the wing, complete
32
M. Freeman./Mech. Dev. 48 (1994) 25-33
loss of rhomboid function does not lead to complete loss of wing vein and Ellipse mutations restore veins lost in a rhomboid mutation without restoring rhomboid expression in the vein primordia. Both these observations suggest that rhomboid is not absolutely required for all vein development. It is possible that the observed low penetrance of rhomboid loss-of-function mutations in the eye is caused by a similar lack of requirement, although the degree of redundancy would have to be greater in the eye since most eye development occurs normally in the absence of rhomboid. Although ectopic expression of rhomboid in the wing and the eye both appear to cause spitz/EGF receptor signalling, different genetic interactions with genes downstream of the EGF receptor are observed in the two tissues. In the wing, mutations in Gap1, which is a negative regulator of Rasl, enhance the effects of ectopic rhomboid, whereas mutations in Sos, an activator of Rasl, do not modify them (Sturtevant et al., 1993). Conversely, in the eye, Sos is a suppressor of ectopic rhomboid and Gapl mutations do not modify the phenotype (this study). As noted above these results do not imply that the components of the pathways are different but they could indicate that the activation of Rasl by the EGF receptor may be subject to slightly different control in different tissues: different members of the pathway appear rate limiting in the wing and the eye. Since signal transduction pathways dependent on Rasl occur in so many developmental contexts, such differences in details of regulation may be important. In none of the cases examined is it clear whether the proposed mediation by rhomboid of the EGF receptor signalling is direct. For example, it could act by interacting with the receptor in the plasma membrane to form an active receptor complex able to bind to ligand or it could act indirectly, for example, by causing increased local signalling by promoting adhesion of abutting cell membranes. Biochemical experiments will be needed to distinguish between these types of model.
3.3. The spitz group in the eye The observation that spitz is required for photoreceptor development indicates that several members of the
spitz group (spitz, pointed and Star) are now known to be required for wild-type eye development; another, rhomboid, may also play a role. It is not clear if this group act as a single pathway in the eye and, indeed, there are some apparent differences. For example, Star and rhomboid are mainly expressed in cells R8, R2 and R5, and Star is only required in those cells (Heberlein and Rubin, 1991; Freeman et al., 1992a; Heberlein et al., 1993). In contrast, pointed, which is a putative transcription factor, is required autonomously in all photoreceptors (M. Freeman, unpublished observation). As noted above, the role of rhomboid in wild-type eye development remains uncertain. The transcript and protein are
expressed specifically early in developing photoreceptors, and clones of rho-/rho- cells are missing a small number of ommatidia (Freeman et al., 1992a). Furthermore, Elp flies that are heterozygous for a null allele of rhomboid have a suppressed phenotype (M. Freeman, unpublished observation), suggesting that near-normal levels of rhomboid are required for EGF receptor signalling in the eye. Nevertheless, the large majority of ommatidia develop normally in the absence of rhomboid so it must be concluded that its function is largely dispensable. This implies that in the developing eye the proposed spitz/EGF receptor interaction does not normally require the mediation of rhomboid. During eye development the EGF receptor is required for at least two steps: proliferation of imaginal disc cells prior to neuronal differentiation and for determination of photoreceptors (Xu and Rubin, 1993). Nothing is known about what ligand, if any, is used for its proliferative function. On the basis of the results presented here, it seems likely that spitz is the ligand during later eye development and that this interaction activates a Rasl dependent signal transduction pathway, via the EGF receptor. This signalling pathway is necessary for photoreceptor determination and may provide the basis for the inductive recruitment of photoreceptors other than R7, in an analagous manner to the specification of the R7 cell fate by the sevenless mediated signal transduction pathway.
4. Experimental procedures 4.1. Fly strains and crosses In order to test for modification of the sev-rho phenotype, each stock was tested against two different sev-rho stocks: sev-rho3 has three copies of the sev-rho transposon on chromosome 3 and sev-rho2 has 2 copies of the transposon, also on chromosome 3. Sev-rho2 and sev-rho3 show a similar phenotype, but the latter has more severe defects. Crosses were carried out in a way which allowed the control flies (sev-rho/+) to emerge from the same vial as the test flies (eg spi/+;sev-rho/+) to ensure that any effect on the severity of the phenotype that could be attributed to growth conditions was equal for test and control animals. Interactions were only scored as positive if a similar effect was scored independently with each sev-rho chromosome. The following people kindly provided me with fly stocks: E. Bier (spitz alleles), U. Heberlein (Star alleles), B. Shilo (Egfr alleles), K. Matthews at the Bloomington Drosophila Stock Center (Eg)Cr alleles), H. Skaer (Egfr alleles), C. K1/imbt (pointed and single minded alleles), M. Simon (Rasl and Sos alleles), Z. Lai and G. Rubin (yan alleles), U. Gaul (Gap1 alleles), E. Hafen (drk, raf, and rolled alleles), J. Vize OCatfacets and groucho alleles), S. Schneuwly (argos alleles) and T. Xu (FRT and fliprelated stocks for producing mitotic clones).
M. Freeman./Mech. Dev. 48 (1994) 25-33
4.2. Scanning electron microscopy and histology Adult heads were prepared for scanning electron microscopy by the procedure described by Kimmel et ai. (1990). Heads were fixed and sectioned as described previously (Freeman et ai., 1992b). 4.3. Mitotic clones and mosaic analysis Mitotic clones of spitz AI4 were produced using the FLP/FRT system as described in detail by (Xu and Rubin (1993). The spitz mutation was crossed onto a chromosome carrying an FR T at chromosomal position 40A. The FLP gene was under the control of the heatshock promoter. The w ÷ gene was used as an autonomous marker for wild-type cells. Clones were induced during the first instar larval stage with 40 min heatshocks at 38°C and were found in 70-80% of flies of the appropriate genotype. Clones were sectioned as described above and phenotypically wild-type mosaic ommatidia were scored for the presence of w+-dependent pigment granules in each of the eight photoreceptors. Only those ommatidia in which all eight cells could be unambiguously scored were included in the analysis.
Acknowledgements I would like to thank Richard Smith for valuable technical assistance; Jeremy Skepper and Tony Burgess of the Cambridge University Department of Anatomy for their help with scanning electron microscopy; and all those people listed in Materials and Methods, who sent me fly stocks. I am grateful to Mariann Bienz, Peter Lawrence, Helen Skaer and John-Paul Vincent for helping to improve the manuscript.
References Baker, N.E. and Rubin, G.M. (1989) Nature 340, 150-153. Baker, N.E. and Rubin, G.M. (1992) Dev. Biol. 150, 381-396. Banerjee, U. and Zipursky, S.L. (1990) Neuron 4, 177-187. Bier, E., Jan, L.Y. and Jan, Y.N. (1990) Genes Dev. 4, 190-203. Brunner, D., Oellers, N., Szabad, J., Biggs, W.H., Zipursky, S.L. and Hafen, E. (1994) Cell 76, 875-888. Campos-Ortega, J.A., Jurgens, G. and Hofbauer, A. (1979) Rouxs Arch. Ent, Org. 186, 27-50.
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Clifford, R. and Schfipbach, T. (1992) Development 115, 853-872. Clifford, R.J. and Schfipbach, T. (1989) Genetics 123, 771-787. Dickson, B., Sprenger, F., Morrison, D. and Hafen, E. (1992) Nature 360, 600-603. Freeman, M., Kimmel, B.E. and Rubin, G.M. (1992a) Development 116, 335-346. Freeman, M., Klfimbt. C., Goodman, C.S. and Rubin, G.M. (1992b) Cell 69, 963-975~ Gaul, U., Mardon, G. and Rubin, G.M. (1992) Cell 68, 1007-1019. Harris, W.A., Stark, W.S. and Walker, J.A. (1976) J. Physiol. 256, 415-439. Heberlein, U., Hariharan, I.K. and Rubin, G.M. (1993) Dev. Biol. 160, 51-63. Heberlein, U. and Rubin, G.M. (1991) Dev. Biol. 144, 353-361. Kimmel, B.E., Heberlein, U. and Rubin, G.M. (1990) Genes Dev. 4, 712-727. Klfimbt, C. (1993) Development 117, 163-176. Kolodkin, A.L., Pickup, A.T., Lyn, D.M., Goodman, C.S. and Bannerjee, U. (1994) Development (in press). Kr~imer, H. and Cagan, R.L. (1994) Curr. Opin. Neurobiol. 4, 14-20. Lai, Z.C. and Rubin, G.M. (1992) Cell 70, 609-620. Lindsley, D.L. and Zimm, G.G. (1992) The Genome of Drosophila Melanogaster. Academic Press, San Diego. Livneh, E., Glazer, L., Segal, D., Schlessinger, J. and Shilo, B.-Z. (1985) Cell 40, 599-607. Mayer, U. and Nfisslein-Volhard, C. (1988)Genes Dev. 2, 1496-1511. Neuman-Silberberg, F.S. and Schiipbach, T. (1993) Cell 75, 165-174. Raz, E. and Shilo, B.Z. (1992) Development 114, 113-123. Raz, E. and Shilo, B.Z. (1993) Genes Dev. 7, 1937-1948. Ready, D.F. (1989)Trends Neurosci. 12, 102-110. Ruohola-Baker, H., Grell, E., Chou, T.B., Baker, D., Jan, L.Y. and Jan, Y.N. (1993) Cell 73, 953-965. Rutledge, B.J., Zhang, K., Bier, E., Yuh, N.J. and Perrimon, N. (1992) Genes Dev. 6, 1503-1517. Simon, M.A., Bowtell, D.D., Dodson, G.S., Laverty, T.R. and Rubin, G.M. (1991) Cell 67, 701-716. Simon, M.A., Dodson, G.S. and Rubin, G.M. (1993) Cell 73,. 169-177. Sturtevant, M.A., Roark, M. and Bier, E. (1993) Genes Dev. 7, 961-973. Tomlinson, A. (1988) Development 104, 183-193. Tomlinson, A., Kimmel, B.E. and Rubin, G.M. (1988) Cell 55, 771-784. Tomlinson, A. and Ready, D.F. (1987) Dev. Biol. 123, 264-275. Wadsworth, S.C., Vincent, W.S. and Bilodaeu-Wentworth, D. (1985) Nature 314, 178-180. Xu, T. and Rubin, G.M. (1993) Development 117, 1223-1237. Zak, N.B. and Shilo, B.Z. (1992) Dev. Biol. 149, 448-456. Zak, N.B., Wides, R.J., Schejter, E.D., Raz, E. and Shilo, B.Z. (1990) Development 109, 865-874.
Mechanisms of Development48 (1994) 25-33
The spitz gene is required for photoreceptor determination in the Drosophila eye where it interacts with the EGF receptor Matthew Freeman* MRC Laboratory of Molecular Biology. Hills Road. Cambridge CB2 2QH. UK
Received 22 April 1994; accepted 24 May 1994
Abstract
Little is known about the mechanisms by which photoreceptors other than R7 are determined during Drosophila eye development. By looking for mutations that modify the phenotype caused by ectopic expression of the rhomboid gene in the eye, I have discovered that the spitz gene is required for photoreceptor determination. Mosaic analysis suggests that spitz, which encodes a TGFc~ homoiogue, produces a diffusible signal during ommatidial development. Other members of the spitz group and the EGF receptor also interact with sev-rho, in a pattern that suggests a model in which rhomboid can act as a mediator of a ligand-receptor interaction between spitz and Egfr in the developing eye. These data suggest that photoreceptors other than R7 use a Ras i signalling pathway activated by the spitz/Egfr interaction, in a manner analagous to the Rasl pathway activated by boss/sevenless in photoreceptor R7. Keywords: Drosophila; EGF receptor; Eye development; rhomboid; Signal transduction; spitz
1. Introduction
The individual facets or ommatidia of the Drosophila compound eye each contain eight photoreceptors, comprising three distinct classes based on their morphology and which opsin they express. Photoreceptors R I - R 6 express the Rhl opsin and have large rhabdomeres (the light gathering organelles); R7 expresses Rh3 or Rh4 and has a small rhabdomere; and R8 also has a small rhabdomere and expresses an unknown opsin. The photoreceptors are determined during the third larval instar in the eye imaginal disc. A morphogenetic furrow sweeps anteriorly across the disc leaving the developing ommatidia in its wake. The photoreceptors in each ommatidium develop in a strict sequence, starting with R8 and finishing with R7, and there is evidence that they are recruited by a series of inductive interactions (see Tomlinson, 1988; Ready, 1989; Banerjee and Zipursky, 1990, for reviews on eye development). The main components of the signalling pathway by which R7 is recruited have recently been identified and they c o m *Tel.: 0223 402234; Fax: 0223 412142.
prise a Rasl dependent signal transduction cascade, initiated by the interaction between the sevenless receptor tyrosine kinase and its ligand, boss (see review by Kr~imer and Cagan, 1994). Much less is known about the molecular mechanisms that are responsible for the determination of the other photoreceptors. However, clonal analysis has shown that although the sevenless tyrosine kinase is not required by any cell other than R7 (Harris et al., 1976; Campos-Ortega et al., 1979; Tomlinson and Ready, 1987), the Rasl protein is essential for the formation of all photoreceptors (Simon et al., 1991). This suggests that a Rasl dependent signalling pathway may be central to all photoreceptor determination but that it is activated by some receptor other than sevenless in the non-R7 photoreceptors. A candidate receptor is the Drosophila epidermal growth factor receptor (Livneh et al., 1985; Wadsworth et al., 1985), also a tyrosine kinase receptor that can activate the Rasl pathway (Simon, et ai., 1991). The EGF receptor is pleiotropic (Clifford and Schiipbach, 1989; Clifford and Schiipbach, 1992; Raz and Shilo, 1992) and is needed for cell proliferation in the eye imaginal disc (Xu and Rubin, 1993), making
0925-4773/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSDI 0925-4773(94)00275-R
26
M. Freeman./Mech. Dev. 48 (1994) 25-33
clonal analysis difficult. However, it does have later functions in eye development: dominant Ellipse alleles of the EGF receptor gene (Egfr) disrupt the number of ommatidia that form (Baker and Rubin, 1989; Baker and Rubin, 1992), and analysis of very small clones indicates that the EGF receptor is probably required in all photoreceptors (Xu and Rubin, 1993). The genes of the spitz group, spitz, rhomboid, Star and pointed, appear to act in a common pathway at several stages in development (Mayer and Nusslein-Volhard, 1988). Recent evidence suggests that this pathway includes the EGF receptor and the spitz group may act 'to mediate EGF receptor signalling. For example, Egfr is required for the establishment of ventral ectoderm as are the members of the spitz group and genetic interactions suggest that they comprise a common pathway (Raz and Shilo, 1993). Similar interactions have been observed in the oocyte and the wing (Ruohola-Baker et al., 1993; Sturtevant et al., 1993). The specific functions of the spitz group genes are not all clear, spitz itself encodes a homolog of the vertebrate EGF receptor ligand, TGFa (Rutledge et al., 1992) and is probably the ligand for the EGF receptor in the ventrolateral signalling pathway, although it is unknown if it acts in a membrane-tethered or a released form. This view is supported by recent biochemical evidence that spitz protein can bind to and activate the EGF receptor in tissue culture cells (R. Schweitzer and B. Shilo, personal communication). The product of the rhomboid gene is predicted to have multiple transmembrane domains (Bier et al., 1990), but is not similar to any known protein (although a similar gene has recently been identified in the nematode by the C. elegans sequencing project - - EMBL accession number Z29117). It is striking that rhomboid is expressed specifically in those cells which are missing in spitz group mutants (Bier et al., 1990), whereas the products of Egfr and spitz are both more widely expressed (Zak et al., 1990; Rutledge et al., 1992; Zak and Shilo, 1992). These observations led to the view that rhomboid may provide spatial cues to the proposed spitz/Egfr interaction and there is recent evidence supporting this model in the oocyte and the wing (Ruohola-Baker et al., 1993; Sturtevant et al., 1993), although the mechanism by which rhomboid might facilitate the interaction is unknown. Star also encodes a transmembrane protein of unknown function (Kolodkin et al., 1994) and pointed encodes two related proteins with ETS domains which are presumed to be transcription factors (Kl/imbt, 1993). In this paper, I examine the role of spitz, the EGF receptor, and other members of the spitz group in the developing eye. Previous work has shown that rhomboid is expressed in a subset of developing photoreceptors and clones of rhomboid mutant tissue show subtle defects in the eye (Freeman et al., 1992a). These defects are most easily interpreted as the occasional failure of om-
matidia to be established in the absence of rhomboid, but the low penetrance of this phenotype has made it difficult to understand the role of rhomboid in eye development. When misexpressed under the promoter of the eye-specific gene sevenless, rhomboid has profound effects: the mystery cells are transformed into photoreceptors and other cells are also affected (Freeman et al., 1992a). These sev-rho flies have a rough eye and I have looked for mutations that modify this phenotype. Mutations in spitz, Egfr, Rasl and another gene in the Rasl pathway, Sos, affect the sev-rho phenotype, supporting the notion that rhomboid can act in an Egfr pathway with the spitz group in the developing eye. Since spitz itself has not previously been shown to act in photoreceptors, I have investigated its role in their development and find that it is necessary for the determination of all photoreceptors. Clonal analysis indicates that it is probably acts as a diffusible factor of rather limited range. I propose that the interaction between spitz and the EGF receptor activates the Rasl dependent pathway necessary for photoreceptor determination in cells other than R7. 2. Results
2.1. Loss of function spitz mutations suppress the sevrhomboid phenotype The eye defects caused by sev-rho (Fig. 1) are dose sensitive; that is, they become more extreme with increasing copy number of the sev-rhomboid transposon (data not shown). This implies that the severity of the defects is sensitive to the degree of signalling mediated by the ectopic rhomboid, which in turn suggests that a 50% reduction in dose of other members of the signalling pathway might significantly alter the phenotype. I have tested many mutations for the ability to suppress or enhance the sev-rho rough eye (Table 1) and those which appear to be involved in the pathway are described below. spitz mutations strongly suppress the sev-rho phenotype in a haplo-insufficient manner (Fig. 1). spitz is a recessive mutation: that is, flies which have only one wild-type copy of the gene are indistinguishable from wild-type flies. In contrast, one wild-type copy of spitz is not sufficient to support normal levels of rhomboid signalling in sev-rho eyes: three loss-of-function alleles dominantly suppressed the sev-rho rough eye, spi r25 and spi AI4 strongly, and spi °Ege more weakly. Sections through such eyes indicate that all the defects caused by sev-rho are equivalently suppressed. This result suggests that in order for sev-rho to change the fates of the mystery cells into photoreceptors and to cause the other defects, spitz gene product is required in nearly wildtype doses: halving the dose of spitz significantly reduces the degree of transformation.
M. Freeman./Mech. Dev. 48 (1994) 25-33
27
Table 1 Mutations tested for genetic interaction with sev-rhomboid Genotype
Interaction with sev-rho
spiAU/+ spiTeJ/+ spiOege/+ SetS/+
S5671/+ EgfrEt/+ EgfrE3/+
0 (additive) 0 (additive)
EgfdI/+
Egfrf2/+ Egfrf4/ +
Fig. !. Mutations in the spitz gene dominantly suppress the sevrhomboid phenotype. A and C show a scanning electron micrograph and a 2 t~m section, respectively, through an eye of a fly carrying three copies of the sev-rhomboid transposon (sev-rho3 flies). The rough eye is caused primarily by the presence of supernumerary photoreceptor cells but also by missing pigment cells and misorientations of the ommatidia. In C, the photoreceptors are identified by their round, dark organelles, called rhabdomeres, which consist of the light trapping membranes; the pigment cells are identified by the refractive pigment granules within them and by the characteristic lattice which they form around the ommatidia. Arrowheads indicate ommatidia with extra photoreceptors. B and D show similar views of a fly of the genotype spiXt4/+;sev-rho3/+, that is one with three copies of the sev-rho transposon and with only 50% of the dose of the wild-type spitz gene product (spitz At4 appears to be close to a null mutation). The reduction in spitz dose has returned the eye to a near wild-type phenotype, with a regular array of ommatidia, the stereotypical trapezoid arrangement of the seven photoreceptors visible in this plane and the unbroken lattice of pigment cells. The rescue is not always complete, but is always unambiguous. In panel D, a slightly misorientated ommatidium is indicated by an arrowhead.
2.2. spitz is required f o r photoreceptor determination spitz is a n e m b r y o n i c l e t h a l m u t a t i o n , s o in o r d e r t o d i s c o v e r w h e t h e r it a c t s in w i l d - t y p e eye d e v e l o p m e n t , I h a v e m a d e c l o n e s o f spi-/spi- t i s s u e in t h e eye b y m i t o t i c r e c o m b i n a t i o n . T h e s e spitz- c l o n e s s h o w severely d i s r u p t e d p h o t o r e c e p t o r d e v e l o p m e n t , t h e m a i n d e fects b e i n g t h e loss o f p h o t o r e c e p t o r s a n d t h e loss o f
Egfrfa/+ Egfrf7/ + Egfrft 3/+ Egfrf24/+ EgfrtOptot/+
-/0
pnte/+ RasletB/+ Rasle2F/+ Sose°O/+
0
-/0
Sose2H/+ Sose4a/+
-/0
),an i/+ yanel+ Gap1 st6/+ Gap1 s:/+ drkSu(Sev)Stt/+
++ +++ 0 0 0
rafm%
o
faf o4/+ fafl ~Os/+ argosgilS/+ argosgilt5/+
0 0 0 0
groFP2/+ simSS/+ simHg/+
0 0 0
rafltU7/+ rlt/+ rltOa/+
0 0 0
Standard cross procedures were used to produce flies that were heterozygous for the mutations shown and which carried two or three copies of the sev-rho transposon (see Experimental procedures). Symbols indicating the results are as follows: 0, no detectable interaction; - - - , strong suppression of sev-rho phenotype; - - , moderate suppression; - , weak suppression; + + +, strong enhancement of sevrho phenotype, + +, moderate enhancement. The nomenclature of the alleles used is as described in Lindsley and Zimm (1992), where possible, and otherwise follows the convention of earlier publications. w h o l e o m m a t i d i a (Fig. 2). spitz- p h o t o r e c e p t o r s a r e f o u n d , i m p l y i n g t h a t n o t all p h o t o r e c e p t o r s n e e d t o m a k e s p i t z in o r d e r t o d e v e l o p n o r m a l l y . S i n c e o m m a t i d i a a t t h e b o r d e r b e t w e e n w i l d - t y p e a n d spitz- tissue c o n t a i n a m o s a i c o f spitz + a n d spitz- cells, it is
28
M. Freeman./Mech. Dev. 48 (1994) 25-33 Table 2 Mosaic analysis of the spitz "414mutation in the eye
Fig. 2. Mitotic clones of spitz- tissue have reduced numbers of photoreeeptors. A white- spitz- clone within a w + spi ÷ background is shown; the clone is seen as the tissue lacking the refractive pigment granules. In the spitz- region, many ommatidia have fewer than normal photoreceptors (examples indicated by arrowheads) and there are a reduced number of total ommatidia as well, compared to the surrounding wild-type tissue. An example of a mosaic, normally constructed ommatidium, of the kind analysed in the mosaic analysis (see Table 2), is labelled to show that cells R3 and R5 are w ÷ (identified by the presence of black pigment granules adjacent to the rhabdomere), while cells RI, R2, R4, R6 and R7 are w-. R8 resides in the basal region of the retina and cannot be scored in this relatively apical section.
p o s s i b l e t o a s k w h i c h cells a r e r e q u i r e d t o b e spitz + t o make a phenotypically wild-type ommatidium. Such a m o s a i c a n a l y s i s s h o w s t h a t t h e o n l y cell t h a t m u s t b e spitz + is t h e R 8 cell, w h i c h is t h e first p h o t o r e c e p t o r t o b e d e t e r m i n e d ( T a b l e 2). T h e r e is a l s o a c l e a r f u n c t i o n in t h e n e x t t w o cells t o b e d e t e r m i n e d , R 2 a n d R 5 , t h o u g h t h e r e q u i r e m e n t is n o t a b s o l u t e . T h e n e x t t w o cells, R 3 a n d R 4 , a l s o a p p e a r t o s h o w a s l i g h t r e q u i r e m e n t t o e x p r e s s spitz a n d t h e l a s t t h r e e cells, R 1, R 6 a n d R7, show no requirement.
2.3. The E G F receptor and genes in its signal transduction pathway interact genetically with spitz and sev-rho in the eye S i n c e s p i t z is p r o b a b l y a l i g a n d f o r t h e E G F r e c e p t o r in t h e e m b r y o a n d t h e E G F r e c e p t o r is r e q u i r e d f o r eye d e v e l o p m e n t , I t e s t e d w h e t h e r Eg)Cr i n t e r a c t s in t h e eye
Photoreceptor
,%w- spi(n = 153)
%w- spi + (n = 86)
RI R2 R3 R4 R5 R6 R7 R8
50 I1 36 20 10 54 47 0.7
44 31 43 34 28 40 42 0
Mosaic analysis was performed in a standard way (for example see Tomlinson et al. 1988), except that clones were induced using the FLP/FRT system instead of by X-ray (see Experimental procedures). Phenotypically wild-type ommatidia that contained both w + and wphotoreceptors were scored and the individual genotype of all eight photoreceptors was noted (see Fig. 2 for example). Two experiments were carried out, the first with spitz AI4 clones, and the second, a control, with wild-type clones. In the first experiment (middle column), 153 phenotypically wild-type ommatidia in spitz- clones were analysed. All but one had w + (and therefore spi +) R8 photoreceptors, indicating that there is essentially an absolute requirement for R8 to be wild-type in order to form a normal ommatidium. The one case where a w- (and therefore spi-) R8 cell was found could be due to a small amount of leakiness in the spi AI4 mutation. This is an EMS allele and has not been characterised. Although it behaves genetically like a null, a Small amount of residual function cannot be ruled out. The w- R8 cell could also indicate that there is a very small probability of producing a normal ommatidium in the absence of spitz in R8, possibly by diffusion of sufficient spitz protein from neighbouring ommatidia (see discussion); or the w- R8 could be an artefact caused by a missing section. Not withstanding this case, it is reasonable to conclude that, in general, R8 needs to be wild-type. It is clear that there are significantly fewer than expected mutant cells R2 and R5 (compare the spi- clone with the control wild-type clone), indicating that there is a partial requirement for spitz in these cells. There is also a suggestion of a slight requirement in cells R3 and R4 (the next pair to become determined) but it is not clear whether the observed differences are significant in this case. In the wild-type control experiment, 86 ommatidia with w ÷ R8 cells were scored to determine whether a minor degree of lineage relationship between R8 and R2/R5 is sufficient to account for the reduced number ofspi- R2 and R5 cells observed in the spitz- clones. As can be seen in the third column, there is a detectable lineage relationship between R2/R5 and R8, indicated by the reduced number of wR2/R5 cells compared to other photoreceptors, when R8 is w + but this reduction is much less than that seen in the spitz experiment. This confirms that the observed requirement for spitz in R2 and R5 is a real effect, not a consequence of the relatedness of these cells with R8.
w i t h spitz, s e v - r h o a n d o t h e r m e m b e r s o f t h e spitz group. I f spitz e n c o d e s a l i g a n d f o r t h e E G F r e c e p t o r in t h e eye, a g e n e t i c i n t e r a c t i o n b e t w e e n t h e m w o u l d b e exp e c t e d . R e d u c i n g t h e level o f b o t h spitz a n d Egfr b y h a l f (ie. flies o f t h e g e n o t y p e spi +/+ Egfr) p r o d u c e d n o d e t e c t a b l e p h e n o t y p e . H o w e v e r , spitz w a s f o u n d t o b e a d o m i n a n t s u p p r e s s o r o f Ellipse alleles o f Egfr (Fig. 3). Although the suppression does not have a dramatic
M. Freeman./Mech. Dev. 48 (1994) 25-33
Fig. 3. Interactions with Ellipse. Scanning electron micrographs of from flies of the following genotypes: A. Eg]'rea!/+. B. spiAU+/+Egfr EBI. The Ellipse B! allele of Egfr causes a dominant rough eye phenotype, A, which is characterised by disorganised ommatidia and a reduction in size of the eye. B. Reduction of the dose of spitz by 50%, by making the fly heterozygous for a spitz loss-offunction mutation causes some suppression of the Ellipse phenotype: this is seen as a significant increase in the overall size of the eye but is most obvious when ommatidia are counted (Table 3).
29
eyes
effect on the morphology of the eye, it is clear when the number of ommatidia are counted (Table 3). Reduction by half of the level of spitz increases the mean number of ommatidia in Elp/+ eyes from 432 to 630 (wild-type eyes have approximately 750 ommatidia). The exact nature of Ellipse mutations is not clear but they are dominant mutations in the EGF receptor and are believed to cause hyperactive signalling by the receptor in its normal cells, rather than ectopic signalling (Baker and Rubin, 1989; Baker and Rubin, 1992; Zak and Shilo, 1992). The individual ommatidia are mainly wild type in Elp eyes, but their number is dramatically reduced. The observation that reducing the amount of spitz protein suppresses the phenotype of these mutations is consistent with spitz being a ligand for the EGF receptor. The model that ectopic rhomboid activates an EGF receptor signalling pathway is supported by the observation that loss-of-function mutations in Egfr dominantly suppress the sev-rho phenotype (Fig. 4B), indicating
Table 3 Spit: suppresses the EIp phenotype Genotype
No. of ommatidia (mean ± S.D.)
EgfreS//+ spiAt4+/+Egfr es!
432 ± 18 (n = 6) 630 + 54 (n = 6)
The total number of ommatidia in eyes of the indicated genotypes were counted. In each case the control flies were grown in parallel to the experimental flies to minimise any variability in growth conditions that might effect the phenotype, spi AI4 behaves genetically as a null allele. The differences are significant at p < 0.001 by two-tailed t-test.
Fig. 4. Further interactions with sev-rhomboid. Eyes of the following genotypes are shown. A. sev-rho3/+. B. Egfr/~/+; sev-rho3/+. C. Rasletb/sev-rho3. D. Sose°d/+;sev-rho3/+. E. S2tS/+;sev-rho3/+. A. As seen in Fig. IA, three copies of the sev-rho transposon causes a rough eye. B. Reduction in the dose of the EGF receptor by 50% significantly reduces the severity of the sev-rho phenotype. Similarly, reduction in the doses of the Rasl gene (C) and the Sos gene (D) also suppress the sev-rho phenotype. E. Reduction in the dose of the Star gene is a very effective suppressor of the sev-rho phenotype: the eyes of these flies are almost completely wild-type. The dominant rough-eye phenotype of Star itself is extremely weak (see Fig. 5A) and is hardly detectable in these flies.
that near-normal levels of Egfr are required for ectopic rhomboid to produce its effects. A range of Egfr- alleles was tested and in most cases the degree of suppression of sev-rho correlated with the severity of the allele (see Table 1). Many of the genes in the Egfr signal transduction pathway have been identified (Simon et al., 1991; Dickson et al., 1992; Gaul et al., 1992; Simon et al., 1993; Brunner et al., 1994) and I have looked for genetic interactions between sev-rho and several of them. Of these genes, loss-of-function mutations in the Rasl gene and the Sos gene also dominantly suppressed the sev-rho phenotype (Fig 4C and D), while no interaction was detected between sev-rho and Gap1, drk, Rafl or rolled. This indicates that Rasl and Sos probably are in the same pathway as rhomboid, as predicted if rhomboid participates in EGF receptor signalling. In contrast to these results, Sturtevant et al. (1993) a found that Gap1 was an effective enhancer of ectopically expressed rhomboid in the wing but that mutations in Sos did not affect the wing phenotype. It should be noted that the lack of an interaction with members of the EGF receptor signalling pathway does not indicate that these components are not involved: this kind of dominant interaction only identifies genes whose products are close enough to
30
M. Freeman./Mech. Dev. 48 (1994) 25-33
Fig. 5. Interactions with Star. Eyes of the following genotypes are shown. A and E. $218/+. B and F. S218/spiAI4. C and G. S218/Egfr f2. D and H. S218/EgfrEnt. The top row of panels show scanning electron micrographs and the bottom row, 2 #m sections through adult eyes. A and E. Star mutations have a weak eye phenotype that varies between undetectable and barely rough; in sections an occasional missing photoreceptor is seen as illustrated by the arrowhead in E. B and F. The Star phenotype is enhanced by a 50% reduction in dose of the spitz gene. F shows that this enhancement takes the form of a higher proportion of ommatidia with missing photoreceptors. C and G. A reduction of the EGF receptor dose also enhances the Star phenotype. D and H. There is a strongly synergistic interaction between Star and Ellipse mutations of the EGF receptor. The eyes of these flies are severely reduced in size and extremely rough. This phenotype is much more severe than a simple addition of the dominant effects of Elp and Star (see Figs 3A and 5A), indicating an interaction between these two mutations. Sections show that there are a greatly reduced number of photoreceptors in all ommatidia.
being rate limiting that a 50% reduction in gene dose reduces the overall signalling level significantly.
2.4. Star acts in the same pathway Star is a n o t h e r m e m b e r o f the spitz g r o u p that interacts with sev-rho in the eye. Star d o m i n a n t l y suppressed all aspects o f the sev-rho p h e n o t y p e very effectively (Fig. 4E). Again, a 50% reduction in Star gene dose leads to a greatly reduced sev-rho phenotype. I have also examined genetic interactions in the eye between Star, spitz a n d Egfr (Fig. 5). H e t e r o z y g o u s Star adults have very slightly rough eyes, but when the dose o f Egfr or spitz is reduced by 50%, the rough eye is enhanced. There is also a synergistic interaction between Star and Ellipse alleles o f Egfr: both give d o m i n a n t rough eyes, but the d o u b l e heterozygote shows a p h e n o t y p e m o r e severe than w o u l d be expected from a simple additive effect. These S +/+ Egfr e eyes are very
rough and reduced in size and sections t h r o u g h them show that most o m m a t i d i a have a reduced n u m b e r o f p h o t o r e c e p t o r s (Fig 5D a n d H). There are also defects in orientation, spacing, a n d pigment cell a n d bristle numbers. It is n o t clear why b o t h loss-of-function a n d gain-of function m u t a t i o n s in Egfr should enhance the Star p h e n o t y p e b u t the degree o f e n h a n c e m e n t is so different that different mechanisms m a y be involved in each case. Nevertheless, the o b s e r v a t i o n that Star shows these genetic interactions with spitz and Egfr further supports the view that these molecules share a p a t h w a y in the eye.
2.5. yan mutations enhance sev-rhomboid The only interaction observed between sev-rho a n d a gene other than Egfr or a m e m b e r o f the spitz g r o u p was with yan, a negative r e g u l a t o r o f p h o t o r e c e p t o r determ i n a t i o n that encodes a potential transcription factor
M. Freeman./Mech. Dev. 48 (1994) 25-33
Fig. 6. Mutations in yah enhance the sev-rho phenotype. The figure shows a scanning electron micrograph, A, and a 2 /zm section, B, through eyes from flies of the genotypeyanP/+;sev-rho3/+. Comparing this phenotypewith that of sev-rho3/+(Figs 1A and 4A), it is clear that there is a considerable increase in the severity of the sev-rho phenotype when the dose of yan is reduced. The section shows that now almost all ommatidiahave 2 or 3 extra photoreceptors.There are also frequent missing pigment cells and ommatidial misorientations, though these defects could be secondary to the extra-photoreceptor phenotype.
with an ETS domain (Lai and Rubin, 1992). Heterozygous yah mutations severely enhance the sevrho phenotype (Fig. 6). Sections through sev-rho/yan eyes show that the extra roughness is caused by an enhancement of all the defects seen in sev-rho/+ eyes. Most ommatidia have between two and four extra photoreceptors, there are many missing pigment cells and no trace of normal orientation or spacing is found. These observations are consistent with the view that yan may act as a repressor of photoreceptor development: when its dose is reduced, the additional photoreceptor recruitment that occurs in sev-rho eyes is greatly increased. 3. Discussion
3.1. The function of spitz in the eye The data presented here show that spitz is necessary for wild-type photoreceptor development. The mosaic analysis indicates that the only cell that must make spitz protein is the first to be determined, R8, and there is a progressively decreasing requirement in the subsequent photoreceptors to produce it. A model arises from this data, coupled with the predicted protein sequence of spitz (Rutledge et al., 1992), which suggests that it may function as a diffusible factor, and the observed requirement for the EGF receptor during photoreceptor determination (Xu and Rubin, 1993). The spitz protein may act as a diffusible ligand which triggers an EGFreceptor-dependent pathway necessary for photorecep-
31
tors to be determined. The observed partial requirement of spitz to be produced by cells R2 and R5 is not easily explained by a model in which a precise interaction occurs between membrane bound ligand and its receptor, but might be expected if the ligand is diffusible. As long as a presumptive photoreceptor is presented with sufficient spitz protein, the cell which produces the signal is not critical; what is important is the local concentration. There would be an absolute requirement for spitz protein to be produced by the first cell to start developing (R8), since without that, the next cells (R2 and R5) could not receive any signal, but after that point there is no requirement for any particular cell to be spitz + as long as there are enough cells to keep the overall level high enough. The partial requirement in cells R2 and R5 suggests that the spitz produced by R8 is not sufficient and as R2 and R5 are also early to develop and, like R8, have a central location in the ommatidium, they are the prime additional source of spitz ligand. It is significant that no normal ommatidia were found in which only R8 was wild-type and that there were a reduced number of ommatidia where only R8 plus one other cell was wildtype (compared to control wild-type clones; data not shown). This suggests that two to three wild-type cells are required to produce a sufficient local concentration of spitz protein for all photoreceptors to develop normally. The mosaic analysis also allows a limit to be set on the range that the spitz signal can diffuse. Since there is essentially an absolute requirement for spitz to be expressed in R8 to make a normal ommatidium, sufficient spitz must not be able to diffuse in from neighbouring wild-type tissue. Therefore spitz protein appears to be able to move between cells within an ommatidium (up to about two cell diameters) but not between ommatidia, which would require a diffusion range of four to five cell diameters. 3.2. Mediation of the Egfr pathway by rhomboid The investigation of rhomboid during oogenesis has led to the conclusion that it encodes a cell autonomous molecule in the membrane that mediates the interaction between the EGF receptor in the dorsal follicle cells and its ligand produced by the oocyte (Ruohola-Baker et al., 1993). There is now convincing evidence that the ligand in that case is the product of the gurken gene which, like spitz, is a molecule similar to TGFc~ (NeumanSilberberg and Schiipbach, 1993). Another study has looked at the role of rhomboid in the developing wing, where it regulates the formation of wing vein(Sturtevant et al., 1993). In that case, rhomboid protein appears to mediate an interaction between the EGF receptor and the spitz protein. Similarly, the ectopic expression of rhomboid in the developing eye activates a spitz/Egfr signalling pathway, providing further evidence that rhomboid acts in this pathway. In the wing, complete
32
M. Freeman./Mech. Dev. 48 (1994) 25-33
loss of rhomboid function does not lead to complete loss of wing vein and Ellipse mutations restore veins lost in a rhomboid mutation without restoring rhomboid expression in the vein primordia. Both these observations suggest that rhomboid is not absolutely required for all vein development. It is possible that the observed low penetrance of rhomboid loss-of-function mutations in the eye is caused by a similar lack of requirement, although the degree of redundancy would have to be greater in the eye since most eye development occurs normally in the absence of rhomboid. Although ectopic expression of rhomboid in the wing and the eye both appear to cause spitz/EGF receptor signalling, different genetic interactions with genes downstream of the EGF receptor are observed in the two tissues. In the wing, mutations in Gap1, which is a negative regulator of Rasl, enhance the effects of ectopic rhomboid, whereas mutations in Sos, an activator of Rasl, do not modify them (Sturtevant et al., 1993). Conversely, in the eye, Sos is a suppressor of ectopic rhomboid and Gapl mutations do not modify the phenotype (this study). As noted above these results do not imply that the components of the pathways are different but they could indicate that the activation of Rasl by the EGF receptor may be subject to slightly different control in different tissues: different members of the pathway appear rate limiting in the wing and the eye. Since signal transduction pathways dependent on Rasl occur in so many developmental contexts, such differences in details of regulation may be important. In none of the cases examined is it clear whether the proposed mediation by rhomboid of the EGF receptor signalling is direct. For example, it could act by interacting with the receptor in the plasma membrane to form an active receptor complex able to bind to ligand or it could act indirectly, for example, by causing increased local signalling by promoting adhesion of abutting cell membranes. Biochemical experiments will be needed to distinguish between these types of model.
3.3. The spitz group in the eye The observation that spitz is required for photoreceptor development indicates that several members of the
spitz group (spitz, pointed and Star) are now known to be required for wild-type eye development; another, rhomboid, may also play a role. It is not clear if this group act as a single pathway in the eye and, indeed, there are some apparent differences. For example, Star and rhomboid are mainly expressed in cells R8, R2 and R5, and Star is only required in those cells (Heberlein and Rubin, 1991; Freeman et al., 1992a; Heberlein et al., 1993). In contrast, pointed, which is a putative transcription factor, is required autonomously in all photoreceptors (M. Freeman, unpublished observation). As noted above, the role of rhomboid in wild-type eye development remains uncertain. The transcript and protein are
expressed specifically early in developing photoreceptors, and clones of rho-/rho- cells are missing a small number of ommatidia (Freeman et al., 1992a). Furthermore, Elp flies that are heterozygous for a null allele of rhomboid have a suppressed phenotype (M. Freeman, unpublished observation), suggesting that near-normal levels of rhomboid are required for EGF receptor signalling in the eye. Nevertheless, the large majority of ommatidia develop normally in the absence of rhomboid so it must be concluded that its function is largely dispensable. This implies that in the developing eye the proposed spitz/EGF receptor interaction does not normally require the mediation of rhomboid. During eye development the EGF receptor is required for at least two steps: proliferation of imaginal disc cells prior to neuronal differentiation and for determination of photoreceptors (Xu and Rubin, 1993). Nothing is known about what ligand, if any, is used for its proliferative function. On the basis of the results presented here, it seems likely that spitz is the ligand during later eye development and that this interaction activates a Rasl dependent signal transduction pathway, via the EGF receptor. This signalling pathway is necessary for photoreceptor determination and may provide the basis for the inductive recruitment of photoreceptors other than R7, in an analagous manner to the specification of the R7 cell fate by the sevenless mediated signal transduction pathway.
4. Experimental procedures 4.1. Fly strains and crosses In order to test for modification of the sev-rho phenotype, each stock was tested against two different sev-rho stocks: sev-rho3 has three copies of the sev-rho transposon on chromosome 3 and sev-rho2 has 2 copies of the transposon, also on chromosome 3. Sev-rho2 and sev-rho3 show a similar phenotype, but the latter has more severe defects. Crosses were carried out in a way which allowed the control flies (sev-rho/+) to emerge from the same vial as the test flies (eg spi/+;sev-rho/+) to ensure that any effect on the severity of the phenotype that could be attributed to growth conditions was equal for test and control animals. Interactions were only scored as positive if a similar effect was scored independently with each sev-rho chromosome. The following people kindly provided me with fly stocks: E. Bier (spitz alleles), U. Heberlein (Star alleles), B. Shilo (Egfr alleles), K. Matthews at the Bloomington Drosophila Stock Center (Eg)Cr alleles), H. Skaer (Egfr alleles), C. K1/imbt (pointed and single minded alleles), M. Simon (Rasl and Sos alleles), Z. Lai and G. Rubin (yan alleles), U. Gaul (Gap1 alleles), E. Hafen (drk, raf, and rolled alleles), J. Vize OCatfacets and groucho alleles), S. Schneuwly (argos alleles) and T. Xu (FRT and fliprelated stocks for producing mitotic clones).
M. Freeman./Mech. Dev. 48 (1994) 25-33
4.2. Scanning electron microscopy and histology Adult heads were prepared for scanning electron microscopy by the procedure described by Kimmel et ai. (1990). Heads were fixed and sectioned as described previously (Freeman et ai., 1992b). 4.3. Mitotic clones and mosaic analysis Mitotic clones of spitz AI4 were produced using the FLP/FRT system as described in detail by (Xu and Rubin (1993). The spitz mutation was crossed onto a chromosome carrying an FR T at chromosomal position 40A. The FLP gene was under the control of the heatshock promoter. The w ÷ gene was used as an autonomous marker for wild-type cells. Clones were induced during the first instar larval stage with 40 min heatshocks at 38°C and were found in 70-80% of flies of the appropriate genotype. Clones were sectioned as described above and phenotypically wild-type mosaic ommatidia were scored for the presence of w+-dependent pigment granules in each of the eight photoreceptors. Only those ommatidia in which all eight cells could be unambiguously scored were included in the analysis.
Acknowledgements I would like to thank Richard Smith for valuable technical assistance; Jeremy Skepper and Tony Burgess of the Cambridge University Department of Anatomy for their help with scanning electron microscopy; and all those people listed in Materials and Methods, who sent me fly stocks. I am grateful to Mariann Bienz, Peter Lawrence, Helen Skaer and John-Paul Vincent for helping to improve the manuscript.
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