Download Function of the Drosophila TGF-a homolog Spitz is controlled by Star

Mechanisms of Development 107 (2001) 13±23
www.elsevier.com/locate/modo
Function of the Drosophila TGF-a homolog Spitz is controlled by
Star and interacts directly with Star
Frank Hsiung a, Eric R. Grif®s a, Amanda Pickup b, Maureen A. Powers a, Kevin Moses a,*
a
b
Department of Cell Biology, Emory University School of Medicine, 1648 Pierce Drive NE, Atlanta, GA 30322-3030, USA
Molecular, Cell, and Developmental Biology, University of California at Los Angeles, 2203 Life Sciences, 621 Young Drive South,
Los Angeles, CA 90095, USA
Received 6 April 2001; received in revised form 15 May 2001; accepted 17 May 2001
Abstract
Drosophila Spitz is a homolog of transforming growth factor a (TGF-a) and is an activating ligand for the EGF receptor (Egfr). It has been
shown that Star is required for Spitz activity. Here we show that Star is quantitatively limiting for Spitz production during eye development. We
also show that Star and Spitz proteins colocalize in Spitz sending cells and that this association is not coincident with the site of translation ±
consistent with a function for Star in Spitz processing or transmission. Finally, we have de®ned minimal sequences within both Spitz and Star
that mediate a direct interaction and show that this binding can occur in vivo. q 2001 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Drosophila; Star; Spitz; TGF-a; Growth factor; Secretion; Development; Signal transduction; EGF; EGF receptor; Retina
1. Introduction
The Drosophila homolog of the EGF receptor (Egfr)
mediates signal transduction during many aspects of development, including regulation of the cell-cycle and patterning during oogenesis, zygotic and imaginal development
(Schweitzer and Shilo, 1997; Freeman, 1998; Nilson and
SchuÈpbach, 1999; Bier, 2000; Carpenter, 2000; Schlessinger, 2000). In the developing eye, Egfr signaling
mediates the speci®cation of the eye itself, cell growth
and survival, the recruitment of cells to the developing
ommatidia, and the induction of development in the optic
lobes of the brain (Xu and Rubin, 1993; Sawamoto and
Okano, 1996; Dickson, 1998; Dominguez et al., 1998;
Huang et al., 1998; Kumar et al., 1998; Spencer et al.,
1998; Chen and Chien, 1999; Kumar and Moses, 2000,
2001; Yang and Baker, 2001).
The ®rst of several positively acting Egfr ligands known
in Drosophila was Spitz, which is a single EGF domain
growth factor homolog in the transforming growth factor
a (TGF-a)/glial growth factor (GGF) family (Aaronson,
1991; Rutledge et al., 1992; Schweitzer et al., 1995). spitz
acts in patterning the dorsal chorionic appendages, the
embryonic cuticle, central nervous system and the imaginal
discs (Mayer and NuÈsslein-Volhard, 1988; Schweitzer et al.,
* Corresponding author. Tel.: 11-404-727-8799; fax: 11-404-727-4717.
E-mail address: [email protected] (K. Moses).
1995; Golembo et al., 1996; O'Keefe et al., 1997; Stemerdink and Jacobs, 1997; Szuts et al., 1997; Wasserman and
Freeman, 1998). In the developing eye, Spitz is required for
the assembly of the ommatidia after the speci®cation of the
founding R8 photoreceptor cell (Freeman, 1994, 1996; Tio
et al., 1994; Tio and Moses, 1997). At least some of these
functions in the developing eye appear to be conserved in
vertebrates (Reh and Cagan, 1994; Bermingham-McDonogh et al., 1996; Neumann and NuÈsslein-Volhard, 2000).
The Drosophila Star protein and another family of
proteins called Rhomboids are required in the Spitz sending
cell for the proper function of Spitz in all stages of development, including the developing eye (Bier et al., 1990;
Heberlein and Rubin, 1991; Freeman et al., 1992; Heberlein
et al., 1993; Kolodkin et al., 1994; Schweitzer et al., 1995;
Sturtevant and Bier, 1995; Queenan et al., 1997; Schweitzer
and Shilo, 1997; Stemerdink and Jacobs, 1997; Freeman,
1998; Guichard et al., 1999; Wasserman et al., 2000). Star
is an integral membrane protein that is expressed in cells
that secrete Spitz and is localized to the early endoplasmic
reticulum (ER) and nuclear envelope (Kolodkin et al., 1994;
Pickup and Banerjee, 1999). However, while Rhomboid is
also an integral membrane protein, it has been localized to
apical patches only (Sturtevant et al., 1996). This difference
in subcellular localization suggests that Star may act at an
earlier point than Rhomboid in the pathway of Spitz maturation. Schweitzer and co-workers showed that Star and
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F. Hsiung et al. / Mechanisms of Development 107 (2001) 13±23
Rhomboid function is required for the signaling activity of
ectopically expressed full-length Spitz (`membrane' or
`mSpitz') but not for the activity of a truncated form of
Spitz that does not require cleavage to release the factor
domain (`secreted' or `sSpitz', Schweitzer et al., 1995).
This suggests that Star and Rhomboid both act in the
production of a functional Spitz signal in the sending cell
and, furthermore, that they may function in the maturation
of the Spitz factor: in protein cleavage, glycosylation, secretion or presentation. In a conceptually similar experiment,
the expression of Drosophila Spitz in a Xenopus animal cap
sandwich assay has been shown to require both Star and
Rhomboid (Bang and Kintner, 2000). The very fact that
Star nulls are haploinsuf®cient dominant mutations with a
rough eye phenotype indicates that Star is genetically dose
sensitive and suggests that Star function may be quantitatively limiting on the Spitz signal during eye development
(Harris et al., 1976; Mayer and NuÈsslein-Volhard, 1988).
Finally, the published subcellular localizations of Star
protein at the EM level and of Spitz by confocal microscopy
suggest that Star may interact directly with Spitz at some
point during its translation, maturation, secretion or presentation (Tio and Moses, 1997; Pickup and Banerjee, 1999).
In this paper, we present data that support the direct interaction of Star with Spitz in vivo in the developing eye. We
show that Spitz and Star proteins colocalize in developing
retinal cells by confocal microscopy and that the quantitative expression of Star can control the quantitative expression of Spitz ± in both loss and gain-of-function Star
genotypes in the developing eye. Furthermore, as previously
reported (Freeman, 1996), we con®rm that ectopic expression of mSpitz has no phenotypic consequence unless
accompanied by the overexpression of Star. We show that
the binding of Star to Spitz is mediated by the 48-residue
domain of Spitz and by a 19-amino-acid segment of Star
(which we call the `growth factor binding domain' or
GFBD) and that these two short protein segments can direct
the colocalization of `cargo' proteins to the same sub-cellular compartment when expressed in HeLa cells. These data
will be discussed in terms of models for the function of Star
protein in Spitz signaling in vivo.
1993). We thus examined the subcellular expression
patterns of both Spitz and Star proteins as well as spitz
RNA in the developing eye by immunolocalization and/or
RNA in situ hybridization and confocal microscopy (see
Fig. 1 and Section 4).
To detect Spitz protein, we used a mouse polyclonal antiserum that was raised against a portion of Spitz that lies Nterminal to the fully processed growth factor-homologous
domain (Tio and Moses, 1997). Thus the Spitz antigen
detected in the experiments described below can only
include precursor forms of the Spitz protein within the
cells that express it ± we are unlikely to be visualizing the
mature secreted factor (`s-Spitz'). We ®nd that Spitz antigen
is found throughout the cytoplasm of the developing photoreceptor cells (Fig. 1A,D, as published in Tio and Moses,
1997). The Spitz protein is granular rather than evenly
distributed and these granules are both perinuclear (Fig.
1D) and located in the apical microvillae (Fig. 1A). Star
protein is somewhat more evenly distributed (Fig. 1B,E,H)
but is most abundant in granules that colocalize with the
Spitz granules (Fig. 1C,F). This is consistent with an early
association of Star with nascent Spitz protein in the ER (as
published by Pickup and Banerjee, 1999) and the maintenance of this association all the way to the site of signal
transmission (the apical microvillae). However, we cannot
draw any ®rm conclusion that Spitz and Star are adjacent
proteins in the ER in vivo from these data alone: while if
they are naturally adjacent, they are likely to be ®xed in
close proximity to each other by the action of the PLP
(2% paraformaldehyde, 0.01 M NaIO4, 0.075 M lysine,
0.037 M NaPO4, pH 7.4) ®xative but the normal subcellular
membranes are probably disrupted by the subsequent washing step that includes triton. To further explore this proposed
direct interaction between Spitz and Star, we have undertaken biochemical experiments described below.
Moreover, we found that spitz mRNA, while showing a
granular distribution, does not colocalize with the Star/Spitz
protein granules (Fig. 1I). This suggests that Star is not
directly involved in spitz translation. The Star antigen
appears to be more abundant than Spitz in these images.
However, this experiment is far from quantitative and we
are reluctant to draw any conclusion from this observation.
2. Results
2.2. Star function is the limiting factor for the quantity of
Spitz antigen in vivo
2.1. Spitz and Star proteins are colocalized within
developing photoreceptor cells
We and others have shown that Star acts genetically
upstream of spitz, that Star is required for the function of
full length (m)Spitz but not a truncated secreted form
(s)Spitz and that Star protein can be detected in the nuclear
envelope and early ER (Schweitzer et al., 1995; Golembo et
al., 1996; Pickup and Banerjee, 1999; Bang and Kintner,
2000). Furthermore, Star has an essential role in the developing eye (Heberlein and Rubin, 1991; Heberlein et al.,
While it was clear from published work that Star is
required for the expression of Spitz, it was not obvious
that the quantity of Star is normally limiting for Spitz
expression. To test this, we examined the expression of
Spitz antigen in the developing eye in conditions of both
loss-of-function for Star and of Star ectopic expression (Fig.
2). In Star mutant (loss-of-function) mosaic clones Spitz
antigen is greatly reduced (Fig. 2A±C). That it is not entirely
absent may be due to the accumulation of some amount of
unprocessed Spitz precursor protein. We observe that Spitz
F. Hsiung et al. / Mechanisms of Development 107 (2001) 13±23
15
Fig. 1. Spitz and Star protein, but not spitz mRNA colocalize in the developing eye. Confocal images of third instar eye imaginal discs showing Spitz and Star
antigens and spitz mRNA as indicated (see Section 4): (A±C) show an apical optical section, (D±F) are mid-level and (G±I) are basal. In (C,F), Spitz is red and
Star is green. In (I), spitz mRNA is red and Star protein is green. Note colocalization (C,F) of most intense level of stain (examples indicated by arrows). Scale
bar in (A) is 10 mm (all panels to the same scale). Anterior right.
protein level in the Star mutant clones was not visibly
reduced in the furrow itself (arrow in Fig. 2A,C), but only
in the later columns of developing ommatidia. It may be that
Star is not limiting for Spitz protein expression in the furrow
itself and that the later loss is due to a reduced number of
differentiating photoreceptor cells in the clone.
We expressed Star ectopically in the developing eye by
inducing mosaic clones with ey:FLP to drive the eye tissue
to consist entirely of recombinant cells (see Section 4).
FRT-mediated recombination is operationally a one-way
process: after mitosis, the two daughter cells are homozygous. Under these conditions, FLPase is expressed in the eye
anlagen continuously. Thus as development proceeds, the
pool of un-recombined cells is progressively depleted. By
the third instar, the eyes are entirely composed of cells
homozygous for one or other of the two chromosomes.
These clones were visualized using negative marking
with b-galactosidase and the cells within these clones are
also homozygous for a Star cDNA driven by an hsp70 gene
promoter. Thus the third instar eye imaginal disc consists of
patches of cells that express b-galactosidase and are wildtype for Star and patches of cells that are negative for bgalactosidase and which express elevated levels of Star
ectopically. When Star protein is thus overexpressed in
mosaic clones, Spitz antigen now appears anterior to the
morphogenetic furrow (Fig. 2D±F). This indicates that the
quantity of Star is normally limiting for the expression of
Spitz ± at least in some parts of the developing eye (i.e.
anterior to the morphogenetic furrow). This must depend
on the observed low levels of spitz mRNA anterior to the
furrow (Tio et al., 1994; Tio and Moses, 1997).
Others have shown that Star loss-of-function mutant cells
die in the developing eye (Heberlein and Rubin, 1991;
Heberlein et al., 1993). Thus the loss of Spitz antigen in
our Star loss-of-function clones (see Fig. 2A±C) could be
trivially due to the apoptotic death of these cells. To test
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F. Hsiung et al. / Mechanisms of Development 107 (2001) 13±23
Fig. 2. Star function controls Spitz antigen. Confocal images of third instar eye imaginal discs containing clones of cells mutant for Star (see Section 4). (A±C)
Star loss-of-function, note that Spitz antigen is reduced in the clone (arrowheads). LacZ negatively marks the homozygous Star 2 cells. (D±F) Star ectopic
expression clone, note that Spitz antigen is ectopically expressed both sides of the furrow (arrowheads). Spitz and LacZ antigens as indicated. Position of the
furrow indicated with arrows. Scale bars in (A) and (B) are 50 mm (panels in the same row to the same scale). Anterior right.
this, we visualized apoptotic cells in Star loss-of-function
clones using TUNEL (Fig. 3A). We ®nd that there is cell
death in these clones, but that it occurs far posterior to the
furrow (about 10 ommatidial columns or 20 h after the
passage of the furrow). Furthermore, we were able to visualize the near-normal expression of several other proteins in
these clones in the region of the furrow: Atonal (Ato in Fig.
3B) as well as Elav and Boss (data not shown). Thus we
conclude that living cells are present within the Star loss-offunction clones for about 1 day after the passage of the
furrow. Furthermore, these cells are suf®ciently vigorous
to express at least three other proteins. Our observation
that they fail to express Spitz is thus not a simple consequence of cell morbidity, but is a differential effect of loss of
Star function. This is consistent with a direct role for Star in
Spitz protein expression.
For further evidence that Star function is normally limiting for Spitz function in vivo, we drove ectopic and elevated
levels of unprocessed full-length or `mSpitz' expression in
the developing eye using an GMR:Gal4 driver (see Section
4). We ®nd that the resulting adult compound eye is indistinguishable from wild-type (Fig. 4A). This suggests that the
quantity of spitz mRNA expression is not normally limiting
for spitz function in the developing eye. We similarly overexpressed Star and this results in a moderate rough eye
phenotype (Fig. 4B). This suggests that the quantity of
Star function may be limiting in normal development.
When we expressed both mSpitz and Star together in the
same animal, we observed a strong synergy (Fig. 4C). We
observed smooth eyes with little or no red pigment and few
mechanosensory bristles. This suggests a de®cit of acces-
sory cells. When we stained the third instar eye imaginal
discs from these animals, we observed that most or all nuclei
in the late developing ommatidial clusters stain for Elav
(Fig. 4D±F). This is consistent with their differentiation as
extra photoreceptor cells and may explain the reduced
number of accessory cells. Indeed, a very similar result
was observed for the function of these proteins in the developing wing (Pickup and Banerjee, 1999). The fact that
elevating levels of mSpitz has no gross effect on the developing eye until Star protein is also elevated is consistent
with a normal limiting function for Star on Spitz expression.
Fig. 3. Star loss-of-function induces late cell death. Confocal images of
third instar eye imaginal discs containing clones of cells mutant for Star
(see Section 4). TUNEL, Ato and LacZ as indicated. LacZ negatively marks
the homozygous Star 2 cells. Position of the furrow indicated with arrowheads. Scale bar in (A) is 100 mm, (B) is to the same scale. Note that
TUNEL shows apoptotic cells in the Star clones ± but only about 10
columns, or 20 h after the passage of the furrow (arrow in A). Also note
that Ato expression is nearly normal in the clones (arrow in B). Anterior
right.
F. Hsiung et al. / Mechanisms of Development 107 (2001) 13±23
17
Fig. 4. The quantity of Star is the limiting factor for Spitz function in vivo: (A±F) the protein(s) named in the lower right has been over-expressed in the
developing eye (see Section 4). (A±C) SEM of adult compound eyes, anterior right. (D±F) Confocal image of the edge of a third instar eye imaginal disc, red ±
Elav (neural nuclei), green ± actin. Overexpression of mSpitz has no effect and is indistinguishable from wild-type (A). Overexpression of Star has a moderate
effect (B). Overexpression of both Star and mSpitz together is synergistic (C). In the double overexpression (C), there is a de®cit of accessory cells resulting in a
smooth eye and an excess of photoreceptor neurons. Close to the furrow (arrowhead in D±F), there is a near normal array of single neurons. However, as
ommatidial assembly proceeds across the disc, excess numbers of neurons are recruited into each cluster (arrows in D±F). Scale bar in (A) is 100 mm (B,C to
the same scale). Scale bar in (D) is 25 mm (E,F to the same scale).
2.3. The EGF homologous `factor' domain of Spitz protein
mediates speci®c binding to Star in vitro
The colocalization of Spitz and Star proteins is consistent
with direct contact between these two proteins in vivo. To
approach this, we tested a series of fragments of Spitz fused
to glutathione-S-transferase (GST) for their ability to bind to
full-length (66 kDa) Star protein expressed in vitro and
labeled with 35S-methionine (see Section 4). We made
seven such protein fusions (SpitzA±GST through SpitzG±
GST, Fig. 5) and ®nd that the SpitzC fragment strongly
interacts with Star and that SpitzF interacts weakly (Fig.
5). However, we found that the weak binding of SpitzF is
not speci®c ± it also binds luciferase and all sub-fragments
of Star tested (see below). Thus we suggest that SpitzC
(residues 71±125) contains the major, high-speci®city binding domain for Star protein. An eighth Spitz fragment
(SpitzH, residues 75±122) is very similar to but slightly
smaller than SpitzC and behaves identically in this assay
(data not shown). SpitzC and SpitzH closely contain the
domain of Spitz, which is cleaved to release the six cysteine
containing EGF homologous `factor' domain (residues 78±
122). Thus our data are consistent with Spitz binding to Star
via the factor domain.
2.4. A 19-amino-acid fragment from Star protein mediates
speci®c binding to Spitz in vitro
We also sought to de®ne the minimal domain within Star
that is responsible for binding to Spitz. We repeated the
binding experiments described above using GST±SpitzC
and a series of seven sub-fragments of Star, 35S labeled in
vitro (called Star1 through Star7, see Fig. 6). Each Star
fragment was tested for binding to GST alone and to
GST±SpitzC. While Star1 and Star2 both bind GST±SpitzC,
they also bind equally well to GST alone. We thus cannot
demonstrate any speci®c SpitzC binding activity in Star1 or
Star2. However, both Star4 and Star5 do bind speci®cally to
SpitzC and these two fragments overlap over a short range
(residues 402±421). To con®rm this location for a speci®c
GFBD, we expressed a smaller fragment: Star7 (residues
390±420). As this fragment is too small to be labeled in
vitro by our protocol, we fused this to yellow ¯uorescent
protein (YFP). We ®nd that this Star7±YFP fusion is speci®cally precipitated by GST±SpitzC (Fig. 6). As a control,
we tested YFP alone and found that it is not precipitated by
either GST±SpitzC or GST alone. Our data are thus consistent with a GFBD within Star that lies between the N-terminal residue of Star5 and the C-terminal amino acid of Star7
± a domain of only 19 residues.
2.5. The factor domain of Spitz and the GFBD of Star can
interact in vivo
Our in vitro experiments (above) suggest that Spitz binds
to Star via the factor domain and that Star binds to Spitz via
the 19-residue GFBD we de®ned. However, in vitro studies
like these may be prone to artifacts and require in vivo
18
F. Hsiung et al. / Mechanisms of Development 107 (2001) 13±23
Fig. 5. GST±Spitz fusion proteins can precipitate labeled full length Star. At the top is a diagram of the structure of Spitz (230 amino-acids in total). The N and
C termini and the signal sequence, mature factor and transmembrane domains are indicated. Below are seven sub-fragments that were fused to GST (A±G, see
Section 4). These were used to precipitate 35S-methionine labeled Star protein (66 kDa) as shown. Also shown are two controls: glutathione±agarose beads
alone (BDS) and glutathione±agarose beads bound to GST alone (GST). Note that fragment `C' strongly precipitates Star (dark shading) and fragment `F' does
so only weakly. However, Fragment `F' binds other targets such as luciferase (data not shown) and we therefore consider this binding non-speci®c. Fragment
`C' does not bind to a control target (luciferase) in this assay.
Fig. 6. GST±Spitz fragment C speci®cally recognizes a single region of the Star protein. At the top is a diagram of the structure of Star (597 amino acids total).
The N and C termini and the trans-membrane domain are indicated. Below are seven sub-fragments (1±7, see Section 4). These fragments were individually
labeled and precipitated with GST±Spitz fragment C (SpitzC) and glutathione±agarose beads complexed to GST alone (GST, as a control) as shown in the gels.
Protein fragment sizes indicated in kDa. Note that as Star7 is fused to YFP, it runs high on the gel (see Section 4). Note that Star fragment 2 is equally
precipitated by both Spitz and GST ± and we therefore consider this binding non-speci®c. Note that fragments 4, 5 and 7 de®ne a minimal sequence that is
speci®cally bound by Spitz fragment C.
F. Hsiung et al. / Mechanisms of Development 107 (2001) 13±23
con®rmation. To approach this, we expressed two readily
detectable `cargo proteins' in tissue culture (HeLa) cells and
then used these to test for the ability of the Spitz factor
domain and the Star GFBD to cause the `cargo' proteins
to colocalize.
The `cargo' proteins were yellow and cyan ¯uorescent
proteins (YFP fused to another fragment to yield YFP±pyruvate kinase (PK) and CFP). When YFP±PK is transfected
into HeLa cells, it is evenly distributed between the nucleus
and the cytoplasm (Fig. 7A). We directed CFP to the
nucleus by adding a nuclear localization sequence from
SV40 virus large T antigen (CFP±nuclear localization signal
(NLS), Kalderon et al., 1984a,b) and when transfected into
HeLa cells, as expected CFP±NLS is nuclear (Fig. 7E).
When the two fusion proteins (YFP±PK and CFP±NLS)
are cotransfected into HeLa cells, YFP±PK remains generally distributed and CFP is nuclear. This shows that the two
19
fusion proteins do not normally bind signi®cantly to each
other in HeLa cells.
We then fused the 48 amino-acid SpitzH fragment
(containing the factor domain) to CFP±NLS to yield CFP±
SpitzH±NLS and as expected, this fusion protein is still
directed to the nucleus when expressed in HeLa cells (Fig.
7B,H). We fused the GFBD containing Star7 fragment to
YFP±PK to yield YFP±Star7±PK and, as expected the
protein remains evenly distributed (Fig. 7D). However,
when CFP±SpitzH±NLS and YFP±Star7±PK are coexpressed in HeLa cells, both the cyan and the yellow ¯uorescent labels colocalize predominantly to the cell's nuclei
(Fig. 7G±I). This suggests that the two proteins bind
together in the HeLa cells' cytoplasm and are then cotranslocated into the nucleus by virtue of the SV40 NLS on the
CFP±SpitzH±NLS partner. Control experiments show that
translocation of the yellow label (YFP) to the nucleus
Fig. 7. The Spitz factor domain can drive subcellular colocalization of a cargo protein via the Star GFBD in vivo. Confocal images of HeLa cells transfected to
express proteins as indicated (see Section 4): (A±C) shows a cell coexpressing YFP±PK and CFP±SpitzH±NLS. Note that YFP±PK is evenly distributed while
CFP±SpitzH±NLS is predominantly nuclear. (D±F) show a cell coexpressing YFP±Star7±PK and CFP±NLS. Note that YFP±Star7±PK is evenly distributed
while CFP±NLS is predominantly nuclear. (G±I) shows a cell coexpressing YFP±Star7±PK and CFP±SpitzH±NLS. Note that now both proteins are predominantly nuclear. Compare the three YFP expressing cells (A,D,G), and note that YFP±PK is predominantly nuclear only when both the Star GFBD and the
Spitz factor domain are available to link the YFP±PK fusion protein to the CFP±NLS fusion protein. Scale bar in (A) is 20 mm.
20
F. Hsiung et al. / Mechanisms of Development 107 (2001) 13±23
requires both SpitzH and Star7 ± either one alone will not
mediate binding (Fig. 7A±F). It is important to note that we
do not suggest that either Spitz or Star are normally nuclear
proteins. We used this colocalization assay only to demonstrate that the Spitz factor domain and the Star GFBD are
capable of signi®cant and stable binding in a living cell.
3. Discussion
Genetic analysis indicated that Star (with rhomboid) may
function upstream of the transmission of Spitz from sending
cells (Schweitzer et al., 1995; Bang and Kintner, 2000) and
Star protein has been shown to be localized to the nuclear
envelope and the early ER (Pickup and Banerjee, 1999).
Taken together, these data suggested to us that Star may
function in the translation, post-translational cleavage,
glycosylation, secretion or presentation of Spitz. To
approach this, we localized Star and Spitz proteins and
spitz mRNA in a series of pair-wise double stains at the
confocal level. We found that Spitz and Star proteins do
colocalize, but that spitz RNA does not. Furthermore Spitz
and Star proteins appear together in granular structures that
are perinuclear as well as apical in the cells. This suggests
that the Spitz±Star interaction persists through much or all
of the secretory pathway.
We found that controlling the quantity of Star directly
affects the quantity of Spitz antigen seen (in loss and gainof-function mosaic clones and by ectopic expression in the
entire eye). In short, less Star results in less Spitz and more
Star in more (and ectopic) Spitz. Consistent with this, we
found that overexpression of mSpitz alone has no phenotypic
effect, but that overexpression of Star does result in a moderate
rough eye suggesting that normally spitz RNA is in excess and
the quantity of the signal is limited by Star. Furthermore, overexpressing both mSpitz and Star together results in a synergistic effect ± and a grossly disordered eye, with a large excess of
photoreceptors and a de®cit of accessory cells. As the main
function of the Spitz/Egfr signal in the eye is to recruit cells to
the developing clusters and specify them as photoreceptor
neurons, this phenotype is consistent with a great increase in
the quantity of this signal. Our immuno-colocalization data
appear to suggest that there is more Star antigen than Spitz
in the developing eye (Fig. 1). However, it is very dif®cult to
draw any conclusions as to the actual relative abundance of
these proteins ± these experiments were not quantitative.
Taken together, all these data suggest that the quantity of
Star protein is the critical limiting factor for the Spitz/Egfr
signal, at least during the normal development of the
compound eye.
We have also used a series of GST-mediated in vitro
binding experiments to de®ne a single region each in
Spitz and Star that mediate their direct interaction (Fig.
8). In Spitz, this 48-residue segment (SpitzH) is virtually
identical to the `factor' domain ± that part of the protein that
contains the six cysteine residues and other features that
show homology to the small diffusible growth factors of
the TGF-a family (MassagueÂ, 1990; Derynck, 1992). In
Star, we have de®ned a 19-amino-acid GFBD responsible
for binding to Spitz. To con®rm these results, we conducted
a test in vivo: we fused SpitzH to CFP as well as a dominant
NLS. This CFP±SpitzH±NLS fusion when expressed in
HeLa cells is directed to the nucleus by virtue of the NLS
and a cyan-colored signal is detected there by confocal
microscopy. We also made a fusion protein in which an
evenly distributed YFP±PK protein was fused to the
GFBD from Star (YFP±Star7±PK). When YFP±Star7±PK
is expressed alone in HeLa cells, the yellow signal is evenly
distributed, but when coexpressed with CFP±SpitzH±NLS,
the yellow signal moves to the nucleus. This `cargo' experiment con®rms that the Spitz factor domain and the Star
GFBD can bind in vivo. Taken together, the in vitro
`GST-pull down' experiments and the in vivo HeLa cell
`cargo' experiments are consistent with a direct interaction
in the living ¯y between the Spitz factor domain and the Star
GFBB. However, neither of these two experiments tested
this interaction in the secretory pathway.
It is interesting to note that the Spitz `factor' domain is Nterminal to the Spitz trans-membrane domain ± and thus
presumed to lie outside of the plasma membrane (or in the
lumen of the organelles of the secretory pathway). The
GFBD in Star lies C-terminal to its trans-membrane domain
and thus would appear to lie on the wrong side of the plasma
or organelle membranes to interact directly with the Spitz
factor domain. However, structural features of Star have led
others to suggest that Star is actually a type II integral
protein, with its C-terminus outside and its N-terminus
inside (Kolodkin et al., 1994). This is therefore consistent
Fig. 8. Binding domains in Spitz and Star. See text. Six conserved cysteine residues in Spitz indicated by asterisks.
F. Hsiung et al. / Mechanisms of Development 107 (2001) 13±23
21
with a direct interaction between the Spitz factor domain
and the Star GFBD in vivo.
In summary, we have shown that Spitz and Star proteins
associate in living cells in the developing Drosophila
compound eye, that Star controls the quantity of Spitz signal
and that these proteins interact via the factor domain in Spitz
and the GFBD (we de®ned) in Star. Our data are consistent
with a role for Star in some stage or stages of Spitz signal
production subsequent to its translation. These conclusions
are very similar to those reached by others for Rhomboid
family proteins (Bier et al., 1990; Freeman et al., 1992;
Sturtevant et al., 1993; Schweitzer et al., 1995; Sturtevant
et al., 1996; Sapir et al., 1998; Guichard et al., 1999; Bang
and Kintner, 2000; Wasserman et al., 2000). While we can
draw no ®rm conclusions from our data, we suggest that Star
may be involved in a complex in the secretory pathway that
acts in the maturation of Spitz. Star could act before Rhomboid as Star has been localized early in the pathway (Pickup
and Banerjee, 1999) and Rhomboid has been localized to the
apical microvillae (Sturtevant et al., 1996) or they may act
together. There is no evidence to suggest that either Star or
Rhomboid are themselves proteases capable of cleaving
Spitz: perhaps they recruit one. Alternately Star may act
as a chaperone to route the pro-Spitz protein correctly
within the secretory pathway or it might be required for
the correct folding of Spitz or it may recruit glycosylation
enzymes. Indeed, our data suggest that Star can interact with
the Spitz factor domain in vitro in conditions in which it
may not be correctly folded. It is interesting to note that
anterior to the furrow, in the developing eye that Star
appears to be quantitatively limiting on Spitz expression.
It may be that Spitz pro-protein that is not correctly routed
or cleaved may be unstable.
While there are several known Rhomboid proteins, Star
appears to be unique in the Drosophila genome. While
homologs of Rhomboid have been detected in vertebrates
(Guichard et al., 1999; Wasserman et al., 2000), we have
been unable to detect any homolog of Star outside of Drosophila, either by searching for similarities to the entire
protein sequence or to the GFBD alone. We have shown
that Star is essential in Drosophila for the activation of an
otherwise inactive growth factor homolog (Spitz). There
may be proteins with similar functions in vertebrates,
which have conserved structure but which are too far
diverged at the primary sequence level to be found with
current computer searching algorithms.
0.1 M NaPO4, pH 7.4, then ®xed at room temperature for
30±45 min in `PLP'. This solution was made fresh before
use. After ®xation, discs were transferred to 0.1 M NaPO4,
pH 7.4, 0.1% Triton X-100 and washed for 1 h at room
temperature. They were then transferred to 0.1 M NaPO4,
pH 7.4, 0.1% Triton X-100, 10% normal goat serum to
block for 15 min. Then discs were transferred to the primary
antibody (dilution optimized for each antibody used) in
0.1 M NaPO4, pH 7.4, 0.1% Triton X-100, 10% normal
goat serum overnight at 48C. Then discs were transferred
to 0.1 M NaPO4, pH 7.4, 0.1% Triton X-100 and washed for
20 min, twice. Then discs were transferred to 0.1 M NaPO4,
pH 7.4, 0.1% Triton X-100, 10% normal goat serum and
washed and blocked for 20 min. Next discs were transferred
to secondary antibody (dilution optimized for each antibody
used) in 0.1 M NaPO4, pH 7.4, 0.1% Triton X-100, 10%
normal goat serum and incubated at room temperature for
3 h. For colocalization, primary antibodies were: mouse
anti-Spitz (Tio and Moses, 1997); rat anti-Star (Pickup
and Banerjee, 1999). Secondary antibodies were: HRP±
donkey anti-rat (ML, Jackson Lab); Cy5-rat anti-mouse
(ML, Jackson Lab). HRP-donkey anti-rat was ampli®ed
using a Tyramide kit (NEN/Life Science Products). In
mosaic clone experiments, b-galactosidase (LacZ) protein
was visualized using either Rabbit anti-b-galactosidase
(Cortex Biochem) or mouse anti-b-galactosidase (Promega)
primary antibodies and FITC±goat anti-rabbit (Jackson
Labs) or Cy5±goat anti-mouse (Jackson Labs) secondaries,
as appropriate. Atonal protein was visualized with rabbit
anti-ATO (Jarman et al., 1994) primary antibody and
Cy5±rat anti-mouse (ML, Jackson Labs) secondary antibody. Elav protein was visualized with rat anti-ELAV
(Developmental Studies, Hybridoma Bank) primary and
FITC±goat anti-rat (Jackson Labs). Boss protein was visualized with mouse anti-Boss (KraÈmer et al., 1991) primary
and Cy5±rat anti-mouse (ML, Jackson Labs) secondary
antibody. For spitz RNA in situ hybridization, digoxigenin-labeled DNA probes were prepared by polymerase
chain reaction (PCR) from a spitz cDNA clone BA3 (Tio
et al., 1994). Hybridization and visualization were as
published (Kumar and Moses, 2001). Apoptotic cells were
detected by TUNEL stain (Intergen Company) using the
manufacturer's protocols. Filamentous actin was visualized
with rhodamine-conjugated phalloidin (Molecular Probes).
Fluorescent images were obtained using a Zeiss LSM510
confocal microscope. Scanning electron microscopy (SEM)
was as previously described (Moses et al., 1989).
4. Experimental procedures
4.2. Genetics and mosaic clone analysis
4.1. Immunolocalization, RNA in situ hybridization and
microscopy
For colocalization of Spitz and Star proteins in the developing eye, imaginal discs from w; hs-S 8/In(2LR)O third
instar larvae raised continuously at 258C were prepared as
previously described (Tomlinson and Ready, 1987). hs-S 8
was as previously described (Kolodkin et al., 1994). Star
loss-of-function clones were obtained from third instar
For immunolocalization, imaginal discs were prepared
and stained largely as described by Tomlinson and Ready
(1987) with minor modi®cations: discs were dissected in
22
F. Hsiung et al. / Mechanisms of Development 107 (2001) 13±23
larvae of the genotype: ey:FLP/1; S 126 FRT40A/
p(conD)25A FRT 40A. p(conD)25A drives general expression of b-galactosidase in the eye disc (Tio and Moses,
1997). Star ectopic expression clones were obtained from
third instar larvae of the genotype: ey:FLP/1; hs-S 8
FRT43D/FRT43D P(arm:LacZ). Star and mSpitz were
misexpressed using UAS-S 4-3 (Golembo et al., 1996) and
UAS-mSpi (Freeman, 1996) driven by GMR:Gal4 (Pignoni
and Zipursky, 1997).
4.3. Protein binding assays
Fragments of Spitz were generated by PCR with restriction enzyme linked primers (BamH1 and EcoR1), ligated in
frame into pGEX-2T vector (Amrad Corp.), and expressed
in BL-21(DE3) competent cells (Stratagene). Spitz fragments are: (A) amino acids 1±50; (B) 28±70; (C) 71±125;
(D) 130±177; (E) 161±195; (F) 178±213; (G) 196±230; (H)
75±122 (see Fig. 5). Star fragments were generated by PCR.
All Star fragments were synthesized with T7 promoters and
Kozak sequences at their 5 0 ends, and stop codons and
poly(A) addition sites at their 3 0 ends. The PCR products
were used directly for in vitro protein synthesis using the
TNT-coupled transcription/translation kit (Promega). Full
length Star is 597 amino acids, Star fragments are: (1)
amino acids 1±210; (2) 140±279; (3) 244±360; (4) 305±
421; (5) 402±522; (6) 422±597; (7) 390±420 (see Fig. 6).
Fragment 7 is too small for in vitro labeling, it was therefore
fused to YFP to increase its size and then treated similarly to
Fragments 1±6. Binding assays were as previously
described (Dai et al., 2000). Protein products were resolved
on 12% SDS polyacrylamide gels and transferred to nitrocellulose membranes before exposing ®lm at 2808C. Equal
amount of GST fusion protein was loaded in each lane,
standardized by Western blot using a goat-anti-GST antibody (Amersham Pharmacia) and equal quantities of 35S in
vitro labeled full length Star was used for each lane in Fig. 5.
Equal amounts of 35S label for each Star fragment was used
for each lane in Fig. 6.
4.4. Cell culture experiment
CFP±NLS and CFP±Spitz fragment H-NLS were cloned
into the pECFP-C1 vector (Clonetech) between BglII and
BamH1 sites. CFP±NLS was made by PCR amplifying the
SV40 NLS sequence with BglII-linked 5 0 primer and
BamH1-linked 3 0 primer. CFP±SpitzH±NLS was made by
amplifying the Spitz fragment with a BglII-linked 5 0 primer,
and a BamH1-linked 3 0 primer that also includes the SV40
NLS sequence. SpitzH was tested and shown to have the
same binding capability to Star as SpitzC in a GST precipitation assay as described above. Truncated PK (residues 15±
430) was PCR ampli®ed with enzyme site linked primers
and cloned into pEYFP-C1 (Clonetech) between BamH1
and Xba1 sites to generate the YFP±PK construct. Star fragment 7 was re-ampli®ed with a BglII-linked 5 0 primer and
BamH1-linked 3 0 primer, and cloned into YFP±PK between
BglII and BamH1 sites to generate the YFP±Star7±PK
construct. HeLa cells (ATCC) were maintained in DMEM
media (Gibco) plus 10% FCS at 378C with 5% CO2 and
transfected with Fugene 6 (Roche) according to the manufacturer's procedure, then incubated overnight. Cells were
®xed in 4% paraformaldehyde and mounted in vectashield
(Molecular Probes).
Acknowledgements
This work was funded by a grant from the US National
Science Foundation (IBN-9807892). We thank N. Arnheim,
U. Banerjee, V. Fuandez, K. Hagedorn, A. Hodel, M. Stallcup, J. Tower and R. Warrior for advice and discussion and
B.-Z. Shilo for Drosophila stocks.
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