Download Characterization of Star and its interactions with

1731
Development 120, 1731-1745 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
Characterization of Star and its interactions with sevenless and EGF receptor
during photoreceptor cell development in Drosophila
Alex L. Kolodkin,1,* Amanda T. Pickup,2,* David M. Lin,1 Corey S. Goodman1 and Utpal Banerjee
2,†
1Howard Hughes Medical Institute, Department of Molecular and Cell Biology, LSA 519, University of California, Berkeley, CA
94720, USA
2Department of Biology, Molecular Biology Institute and Brain Research Institute, University of California, Los Angeles, CA 90024,
USA
*These two authors have contributed equally to this project
†Author for correspondence
SUMMARY
Loss-of-function mutations in Star impart a dominant
rough eye phenotype and, when homozygous, are
embryonic lethal with ventrolateral cuticular defects. We
have cloned the Star gene and show that it encodes a novel
protein with a putative transmembrane domain. Star transcript is expressed in a dynamic pattern in the embryo
including in cells of the ventral midline. In the larval eye
disc, Star is expressed first at the morphogenetic furrow,
then in the developing R2, R5 and R8 cells as well as in the
posterior clusters of the disc in additional R cells. Star
interacts with Drosophila EGF receptor in the eye and
mosaic analysis of Star in the larval eye disc reveals that
homozygous Star patches contain no developing R cells.
Taken together with the expression pattern at the mor-
phogenetic furrow, these results demonstrate an early role
for Star in photoreceptor development. Additionally, lossof-function mutations in Star act as suppressors of R7
development in a sensitized genetic background involving
the Son of sevenless (Sos) locus, and overexpression of Star
enhances R7 development in this genetic background.
Based on the genetic interactions with Sos, we suggest that
Star also has a later role in photoreceptor development
including the recruitment of the R7 cell through the
sevenless pathway.
INTRODUCTION
question as to whether they are mutations in the same gene. A
role for Star during embryonic development was first
suggested by Mayer and Nusslein-Volhard (1988), who
showed that this gene is involved in dorsoventral pattern
formation in the embryo. Based on their phenotypic similarities, a group of genes including Star (S), spitz (spi), rhomboid
(rho) and pointed (pnt) were called the 'spitz group' genes.
Mutations in any of these loci cause embryonic lethality and
share similar pattern defects in the derivatives of the ventral
ectoderm. In addition to cuticular defects (Kim and Crews,
1993), mutant embryos also display severe central nervous
system (CNS) abnormalities. These defects are caused by the
improper positioning or development of the cells along the
ventral midline of the embryo. In the case of rho, spi and S,
the anterior and posterior commissures are fused, owing to the
failure of the midline glial cells to differentiate and migrate
properly (Klämbt et al., 1991). Star and other members of the
spitz group genes are also involved in the development of the
female germ line and in the formation of a subset of the sensory
organs of the peripheral nervous system (PNS) (Mayer and
Nusslein-Volhard, 1988; Rutledge et al., 1992; Bier et
al.,1990).
The discovery that spitz encodes an EGF-like molecule has
Signal transduction pathways have been shown to mediate
many determinative events that underlie development. Cell fate
changes and directed cell movements often require intercellular and intracellular signaling events in order to enact developmental programs. Analysis of these pathways in Drosophila
and Caenorhabditis elegans has resulted in the identification
and detailed characterization of several molecules that function
in signal transduction events during development (reviewed in
Greenwald and Rubin, 1992). Genetic analysis affords the
opportunity to identify additional members of these pathways,
and we describe here the involvement of the Star gene in two
different signaling pathways in Drosophila eye development.
The Star mutation was isolated by Bridges and Morgan
(1919) on the basis of its dominant rough eye phenotype. The
regular array of facets in the eye was found to be disrupted.
Subsequently, Lewis (1945) described the chromosomal region
that includes Star and the neighboring asteroid rough eye
mutation. Star and asteroid mutations show strong interactions, fail to complement one another for the eye phenotype,
map extremely close to each other, and yet are separable by
rare recombination events. It therefore remained an open
Key words: Drosophila melanogaster, eye development, spitz group
genes, neuronal determination, embryonic midline
1732 A. Kolodkin and others
led to the suggestion that the spitz group genes may participate
in a signaling pathway that includes the Drosophila EGF
receptor (Egfr) (Rutledge et al., 1992). In fact, lethal loss-offunction alleles, Egfrflb, have ventrolateral defects that are
hallmarks of the spitz group genes (Schejter and Shilo, 1989).
In addition, dosage-sensitive interactions between other Egfr
alleles and the spitz group genes have been found. For
example, the wing phenotype of the viable rhoVE mutation is
suppressed by EgfrElp alleles (Sturtevant et al., 1993), and Star
enhances the wing phenotype of Egfrtop mutations (J. Price and
T. Schüpbach, personal communication). Here we present
results that are consistent with the idea that Star and Egfr participate in a common signal transduction pathway.
While the Drosophila embryo provides an ideal system for
the analysis of mutants affecting pattern formation, the
compound eye offers some advantages for studying genes that
have a wide spectrum of developmental functions. This is due
to the ease with which developmental events can be studied at
the level of identified cells and the availability of sensitized
genetic systems, allowing for the functions of these genes to
be separated into distinct pathways (Rogge et al., 1991; Simon
et al., 1991; Dickson et al., 1993). Two signal transduction
pathways affecting photoreceptor cell development in the eye
have been studied in great detail (reviewed in Banerjee and
Zipursky, 1990; Rubin, 1991). One involves the Sevenless
tyrosine kinase receptor (Hafen et al., 1987; Banerjee et al.,
1987a) and the other the EGF tyrosine kinase receptor. The
Egfr gene product is expressed in developing eye discs (Zak
and Shilo, 1992; Baker and Rubin, 1992) and the proper
expression of this gene is important for determining the
spacing of the neuronal clusters (Baker and Rubin, 1989). Each
of these clusters contains eight photoreceptor cells (R cells)
called R1-R8. The clusters develop at a morphogenetic front,
called the furrow, and are spaced at fixed distances, ensuring
the final development of a smooth eye with the facets (also
called ommatidia) in regular hexagonal arrays. In dominant
Ellipse (EgfrElp) mutants, this regular spacing is disrupted
(Baker and Rubin, 1992).
The photoreceptors R8, R2 and R5 are the first cells to differentiate from amongst a large rosette of cells (Wolff and
Ready, 1991). This is followed by the pairwise differentiation
of R3 and R4, then R1 and R6. The R7 cell is the last photoreceptor to differentiate in each cluster (Tomlinson and
Ready, 1987). Detailed molecular and genetic analysis has
established that the neuronal fate of R7 is induced by the previously determined R8 cell. This involves the signaling
molecule, Boss, which is expressed on the surface of R8, and
the tyrosine kinase receptor Sevenless, expressed on the R7
precursor membrane (Reinke and Zipursky, 1988; Hart et al.,
1990; Kramer et al., 1991; Cagan et al., 1992). Sevenless
protein is expressed, but not activated, in cells other than R7
(Banerjee et al., 1987b; Tomlinson et al., 1987; Van Vactor et
al., 1991). The Sevenless and EGF signaling pathways share
many downstream components including Ras1 and Sos (Simon
et al., 1991; Bonfini et al., 1992).
Mosaic analysis shows that Star functions in R cell development (Heberlein and Rubin, 1991), since homozygous
patches of Star alleles produce scars in the adult eye. Interestingly, the ommatidia along the mosaic patch boundary that have
followed a normal course of development require wild-type
Star function in the R2, R5 and R8 cells. Thus, the function of
Star is only required in these three early differentiating cells
for the ommatidium to develop normally.
In this paper, we report the molecular characterization of the
Star gene, its expression in the embryo and a detailed analysis
of its role in photoreceptor cell development. Based on various
genetic interactions, the spatial expression pattern of the Star
transcript, and a clonal analysis of Star in the eye disc, we
conclude that normal Star function is important for the early
signaling processes that cause R2, R5 and R8 to develop and
also for the later Sevenless-mediated pathway that promotes
the development of the R7 cell.
MATERIALS AND METHODS
Screen for second site suppressors of SosJC2
sevE4/Y; SosJC2/SosJC2 males were mutagenized with X-irradiation
(Grigliatti, 1986) and mated to sevE4/sevE4;CyO/Tft females. A
primary screening was done in which flies were allowed to choose
between green and UV light (Banerjee et al., 1987a). In this test, suppressors of SosJC2 with no R7 cells are expected to choose visible
light. However, owing to the incomplete penetrance of suppression in
sevE4;SosJC2 flies, an 8% level of false positives were found amongst
all the mutagenised flies. This method was used as a prescreen to
enrich for mutants. All flies choosing visible light were individually
mated to sevE4; Cyo/Tft females. Their progeny were then screened
by the deep pseudopupil method (see Banerjee et al., 1987a) to look
for true second site suppressors of R7 development.
Mapping of S104E
S104E was mapped meiotically using the multiply marked chromosome al,dp,b,pr,c,px,sp (obtained from the Indiana Stock Center). The
lethality was further mapped using a series of deficiencies (also from
the Indiana Stock Center) in the region between al and dp.
Star P allele
The enhancer trap line SF126 (which was obtained from Y. Hiromi)
contains a P element, inserted at the cytological location 21E1-2. This
P element is designated FZ and includes the E. coli lacZ reporter gene
(Mlodzik and Hiromi, 1992). The transposon was mobilized by
crossing to flies carrying a stable source of transposase activity
(Robertson et al., 1988) and identifying excision events by loss of the
rosy+ marker. These lines were tested for reversion of the Star
phenotype and for presence of the ast phenotype.
cDNA isolation, sequence analysis and northern analysis
A phage genomic library was made in λDash (Stratagene) using DNA
from the SF126 line and was subsequently screened using a P-elementspecific probe to isolate flanking genomic DNA. This was used to
construct a genomic walk of wild-type DNA of approximately 35 kb.
A genomic fragment ~3 kb from the site of the P-element insertion
was used to screen 1×106 clones from a Drosophila embryonic cDNA
library (Zinn et al., 1988). 20 clones were recovered and one 4 kb
clone (called K2C) was sequenced using the dideoxy chain termination method (Sanger et al., 1977) and Sequenase (US Biochemical
Corp.). Templates were made from M13 mp10 vectors containing
inserts generated by sonication of plasmid clones. K2C was completely sequenced on both strands; oligonucleotides and double strand
sequencing of plasmid DNA (Sambrook et al., 1989) were used to fill
gaps. The predicted protein sequence was analyzed using the
FASTDB and BLASTP programs (Intelligenetics). For northern
analysis, total RNA was prepared from staged Drosophila embryos,
loaded (40 µg/lane) onto formaldehyde-containing 1% agarose gels
Star and its interactions with sevenless and EGF receptor in Drosophila 1733
and blotted onto GeneScreen Plus membranes. The filters were subsequently hybridized with 32P-labeled probes made from the Star
cDNA, K2C.
under these conditions recombination events are occuring at a rate of
about 80% of the above genotype.
Construction of Star hsp70-cDNA vector and rescue
experiment
A 2865 bp SnaB1-DraI restriction fragment from K2C, containing the
entire 1796 bp Star ORF, 347 bp of 3′ untranslated sequences, and
722 bp of 5′ untranslated sequences was cloned into a modified Bluescript (Stratagene) vector that allows for the Star sequences to be
isolated as a single KpnI restriction fragment. This KpnI fragment was
cloned into the Carnegie 20-derived hsp70 vector HT-4 (Schneuwly
et al., 1987) and transformed into flies (Spradling, 1986) to give the
Shs.8 and Shs.7 lines.
To assay rescue of Star by the hsp70 cDNA construct, the Shs.8
insertion was first localized to 47A by standard chromosome in situ
hybridization (Ashburner, 1989). S104E,dp,Shs.8 recombinant chromosomes were produced by mating S104E,dp,b, pr/SM6a flies to al,
Shs.8/Cyo flies. Recombinant males were identified by backcrossing
to a recessively marked chromosome; al,dp,b,pr,c,px,sp and 15 independent recombinants were balanced over SM6a. Brother, sister
matings of S104E,dp,Shs.8/SM6a flies from each line were made to
generate flies of a S104E,dp,Shs.8/S104E,dp,Shs.8 genotype. These flies
were viable and had normal eyes, indicating a rescue of S104E by Shs.8.
All recombinant chromosomes were crossed to S104E and ast1 to
confirm the presence of Star. The recombinant chromosomes were
all lethal over S104E and had mild rough eyes over ast1. The presence
of Shs.8 on the recombinant chromosome was determined by scoring
for the linked ry+ marker on the hsp70-cDNA insertion, and by its
ability to enhance R7 development in a sevE4/Y; SosJC2/+ background.
An analagous genetic scheme was used to recover ast1,dp,Shs.8 flies.
The presence of ast1 on the recombinant chromosomes was confirmed
by backcrossing to ast1; in all 17 cases this gave a rough eye. Two
copies of the hsp70 K2C construct rescued the ast1/ast1 eye
phenotype.
RESULTS
Star alleles can be isolated in a sensitized genetic
screen
Mutations affecting the development of the R7 cell in the eye
can be detected in any one of several sensitized genetic backgrounds (Simon et al., 1991; Bonfini et al., 1992; Dickson et
al., 1993). Our screen utilizes a dominant mutation in the Son
of sevenless (Sos) gene, called SosJC2, which partially suppresses the mutant phenotype of the sevE4 allele of sevenless
(Rogge et al., 1991; Bonfini et al., 1992). In sevE4/Y; SosJC2/+
flies, about 16% of the ommatidia have R7 cells, in contrast to
0% in sevE4 alone. The number of R7 cells developing in this
genetic background is sensitive to the dosage of other genes in
the pathway. As shown in Table 1, loss of one copy of boss or
Ras1, genes participating upstream and downstream of Sos
respectively, completely eliminates this suppression (Bonfini
et al., 1992). Similarly, the loss of one copy of a gene encoding
a molecule with an inhibitory function, the GAP1 gene (Gaul
et al., 1992), causes a larger percentage of the ommatidia to
develop R7 cells in this genetic background (Rogge et al.,
1992). The effect of the loss of one copy of other genes on the
suppression level in sevE4/Y; SosJC2/+ flies allowed us to
devise a screening technique to identify suppressors and
enhancers of R7 formation in this double mutant background.
One of the mutants isolated in such a screen was called
104E. When one copy of 104E is placed in a sevE4/Y; SosJC2/+
background, the percentage of ommatidia with R7 cells (the
Table 1. Mutations in genes belonging to the Sevenless
pathway can be detected as suppressors or
enhancers of SosJC2
sevenless enhancer-driven Star cDNA
A 4 kb EcoRI fragment containing the entire Star cDNA was cloned
into the Bluescript vector. The insert was excised as a KpnI-NotI
fragment and then cloned into the pBD365 vector (a gift from Barry
Dickson and Ernst Hafen) which has two sevenless enhancers linked
to the hsp70 promoter and this plasmid was transformed into flies.
Eleven independently isolated transformant lines were named sEStar1 through sE-Star11.
Genotype
Whole-mount in situ analysis of embryos and eye discs
Whole-mount in situ analysis of Star transcripts in both embryos and
eye discs was performed essentially as described (Kopczynski and
Muskavitch, 1992), with minor modifications to increase sensitivity
(Kopczynski and Goodman, unpublished data).
Individual ommatidia were scored for the presence of R7 by using the
optical technique of pseudopupil (Franceschini and Kirshfeld, 1971). boss1 is
a null allele of boss (Hart et al., 1990). The deficiency, Df (3R)by10, which
includes Ras1 was used. Gap1sxt is a P allele of the Gap1 gene called sextra
(Rogge et al., 1992).
Generating flp patches in the larval eye disc
To produce patches of homozygous S1 in the eye disc (Golic, 1991),
virgins from a stock with an FRT element inserted at 33, proximal to
Star, were crossed to males from a third chromosome FLP stock (with
the FLP recombinase on the Mkrs balancer chromosome, marked with
Sb). Female progeny, of the genotype FRT/+; FLP, Sb/+ were mated
with males from a recombinant S1, FRT/SM6a line. 15-36 hours after
egg laying, larvae from this cross were heat shocked in a waterbath
for 90 minutes at 38.5°C. Eye discs from late third instar larvae were
dissected out and stained with mAb22C10 (provided by Seymour
Benzer) as described in Kramer et al. (1991). From this cross, 1/8th
of the progeny are expected to have the genotype FRT/ S1, FRT; Flp,
Sb/+, which allows a FLP-induced recombination event to occur. 600
discs were stained and 60 of them contained patches. We estimate that
sevE4/Y; SosJC2/+
sevE4/Y; SosJC2/+; boss1/+
sevE4/Y; SosJC2, +/+, Df(Ras1)
sevE4/Y; SosJC2/+; Gap1sxt/+
Ommatidia with
central cells (%)
Total ommatidia
counted
16
0
0
49
2392
2337
2180
1934
Table 2. Star and asteroid mutations are suppressors
of SosJC2
Genotype
sevE4/Y; SosJC2/+
sevE4/Y; S104E, SosJC2/+, +
sevE4/Y; +, SosJC2/S54, +
sevE4/Y; +, SosJC2/S11N23, +
sevE4/Y; +, SosJC2/S1, +
sevE4/Y; +, SosJC2/ast1, +
sevE4/Y; +, SosJC2/ast4, +
Ommatidia with
central cells (%)
Total ommatidia
counted
16
0
0
0
0
1
3
2392
2128
2312
1980
2010
2234
1678
S1, S54 and S11N23 are presumed loss-of-function alleles of Star; ast1 and
ast4 are loss-of-function asteroid alleles.
1734 A. Kolodkin and others
level of suppression) drops from the 16% found in sevE4/Y;
SosJC2/+ flies to 0% (Table 2). The 104E mutation has a
dominant rough eye. This dominant phenotype was mapped by
standard recombination between al and dp on the 2nd chromosome. 104E is homozygous lethal and the lethality maps to
the same region as the dominant rough eye phenotype. The
map position and phenotypes are consistent with 104E being a
mutant allele of the Star gene. This was confirmed by standard
complementation tests; 104E is completely lethal when placed
over the S54, S11N23 and S1 alleles of Star. When these alleles
of Star were placed into the sensitized background, they also
lower the suppression level (Table 2). The asteroid mutation
Fig. 1. Genetic interactions of Star with asteroid, Sos and Egfrflb. (A-D and I-L) Scanning electron micrographs (SEMs); bar=66.7 µm.
(E-H and M-P) Light microscope sections (bar=1 µm), of adult eyes. (A,E) Wild type. The regular hexagonal array of facets is seen. The dark
structures are rhabdomeres of the photoreceptor (R) cells. At this level of section, rhabdomeres of outer R1-6 cells surround the central R7
rhabdomere. The rhabdomere of R8 is proximal to R7 and is therefore not seen in this section. (B,F) S1/+. The regular arrangement of facets is
disrupted, resulting in a slightly rough eye. In sections, most of the ommatidia appear wild type, with only occasional examples of ommatidia
that are missing R cells. (C,G) ast1/ast1. The eye is quite rough and smaller than in Star/+. In sections, most ommatidia are found to be wild
type, with a few cases in which the R cells are missing. (D,H) S1/ast1. The eye is very reduced in size, containing few facets. The defects in
external morphology, and also in ommatidial organization, are much more severe than in S1/+ or ast1/ast1 alone. The majority of ommatidia are
missing one or more R cells. Ommatidia are often separated by islands that contain only pigment cells. (I,M) S54/+. The phenotype is similar
but slightly weaker than S1/+. (J,N) S54, +/+, Sosx122. The eye is slightly rougher than S54/+. The section shows that many more ommatidia are
missing central R7 cells and/or outer R1-6 cells than in S54/+. Also see Table 3. (K,O) flb2L65/+. The eye looks wild type, both externally and in
sections. (L,P) +, Egfrflb/S54, +. The eye phenotypes of S54/+ and Egfrflb/+ are synergistically enhanced in this double mutant combination.
Externally, the eye is rougher than either S54/+ or Egfrflb/+. In sections, many ommatidia have abnormal numbers of R cells (see text for
Star and its interactions with sevenless and EGF receptor in Drosophila 1735
was placed into this sensitized genetic background. The ast1
and the ast4 mutations lower the suppression level to 1 and 3%
respectively. Thus, in the SosJC2 suppression paradigm, ast
alleles have the same effect as Star (Table 2).
Adult eye phenotype of Star and asteroid
A dominant rough eye phenotype is seen in Star (compare Fig.
1A and B). This external roughness is likely to be largely due
to defects in non-photoreceptor cells, like cone and pigment
cells. Additionally, the weak external roughness is reflected in
rather mild defects in photoreceptor organization. We analyzed
these defects further by cutting sections tangential to the
ommatidia. As shown in Fig. 1F, most of the ommatidia are
wild type. In a small, but reproducible fraction of ommatidia,
either R7 or the outer R1-R6 type cells fail to develop (Table
3). All asteroid mutations are viable and recessive, and have
rough eyes with minor defects in ommatidial assembly (Fig.
1C,G). In S−/ast− transheterozygotes, the S−/+ eye phenotype
is greatly enhanced; the eyes are reduced in size and sections
show that many more ommatidia are missing R cells (Fig.
1D,H).
Genetic interactions suggest a role for Star in R cell
development
As discussed above, S−/+ flies have mild disruptions in their
ommatidial organization. Table 3 shows that, while one copy
of a Sos loss-of-function mutation, Sosx122/+, shows no
phenotype, in a S−, Sos+/S+, Sosx122 fly, significantly increased
numbers of ommatidia are missing R cells as compared to S−
alone (Fig. 1M,N; Table 3). As shown in Table 3, 45% of the
ommatidia in these flies are defective. Of these, 21% of the
ommatidia fail to develop R7 cells, even though these flies are
wild type for the sevenless and boss loci and 40% of the
ommatidia lack one or more of the outer R1-6 cells. At basal
planes of section, an R8 cell is found to develop normally in
each ommatidium (not shown). In summary, Star not only
interacts with the gain-of-function allele (SosJC2) in a sevenless
background, but also with a loss-of-function allele (Sosx122) in
an otherwise wild-type background and, in each case, the
development of R7 cells is affected. In addition, an effect on
the development of R1-6 can be seen in the interaction with
the loss-of-function allele of Sos.
To study further the role of Star in eye development, we
combined Star with mutations in the Drosophila EGF receptor.
We detected a synergistic interaction in the eye between strong
alleles of Star and the gain-of-function allele EgfrElp (Fig. 2).
When compared with an Elp1/+ eye (Fig. 2B) or a S1/+ eye
(Fig. 2A), flies of a S1,+/Elp,+ genotype have a dramatically
reduced eye containing only a small number of facets (Fig.
2C). Furthermore, when the Elp allele is combined with one
copy of a chromosome that is deleted for both Star and
asteroid, Df(2L)ast 2, the reduced eye phenotype is much more
pronounced (Fig. 2E). These results show that the EgfrElp
mutation interacts dominantly with loss-of-function alleles of
both Star and asteroid. As shown in Fig. 2F, no interaction is
seen in the eye when Elp is combined with one copy of Shs.8
in which the Star cDNA is overexpressed using the heat shock
promoter (see below).
In addition to the interactions between Star and Ellipse, we
also observed dominant interactions in the eye between Star
and loss-of-function mutations in Egfr. In combination with
Egfrflb alleles, we observed a significant enhancement of the S−
/+ phenotype. Over 40% of the ommatidia in S, +/+, Egfrflb
flies contain less than the wild-type complement of R cells
(Figs 1O, P). This is a synergistic effect, since only 9% of the
ommatidia are affected in Star/+ and all Egfrflb/+ ommatidia
are wild type. This suggests that Star functions with the EGF
receptor in determining the early events of R cell development.
The above results are consistent with those from previous
studies suggesting that Star and the EGF receptor function
together in the development of a variety of tissue types (Sturtevant et al., 1993; J. Price and T. Schüpbach, personal communication). The involvement of Star in the Sevenless and EGFR
pathways is consistent with the observations of Heberlein et al.
(1993), who have shown that Star interacts with Ras 1.
Cloning the Star gene
During the course of a mutagenesis designed to identify
mutations that affect the embryonic CNS, a lethal P-element
insertion (designated SF126) exhibiting defects characteristic of
the spitz group genes was isolated. Embryos homozygous for
SF126 exhibit CNS and cuticle defects characteristic of Star
mutations, including deletion of medial portions of the denticle
bands and a narrowing of the ventral nerve chord. Standard
complementation tests were performed using several Star
alleles, establishing SF126 as an allele of Star (data not shown).
The cytological location of the insertion in SF126 is 21E1-2, the
location of the Star locus. We confirmed that the SF126
phenotype is due to the P-element insertion by mobilizing it
(see Materials and Methods) and isolating ry− revertants. Out
of 22 revertants, 7 were S+, 14 were S− (exhibiting both eye
and embryonic S phenotypes), and one was phenotypically ast.
Taken together, these data show that SF126 is an allele of Star.
To identify the Star transcript, genomic DNA flanking the
SF126 insertion was isolated and used to conduct a genomic
walk encompassing about 35 kb (see Materials and Methods).
Table 3. Star interacts with a loss-of-function mutation in Sos
Genotype
sev+/Y; S54/+
sev+/Y; Sosx122/+
sev+/Y; +, Sosx122/S54, +
Ommatidia with one
or more outer
cells (R1-R6)
missing, but
with normal R7 (%)
Ommatidia
with R7
missing, but
with normal
R1-R6 (%)
Ommatidia
with both R7
and one or more
outer (R1-R6) cells
missing (%)
Total % of
defective
ommatidia
Total number
of ommatidia
counted
7
0
24
2
0
16
0
0
5
9
0
45
951
1218
517
Sosx122 is a lethal loss of function allele of Sos (Rogge et al., 1991).
1736 A. Kolodkin and others
Genomic sequences were in turn used to probe northern blots,
identifying a 4 kb transcript contained within this walk (Fig.
3C). The SF126 insertion is located within the downstream
intron of this transcription unit (Fig. 3C), defining this transcript as a good candidate to encode the Star protein. The
expression of this transcript is temporally regulated during
embryonic development (Fig. 3B), showing peak expression
between 10 and 14 hours after egg laying. The onset of Star
expression is consistent with the expected temporal requirements for the Star protein as deduced from the observed cuticle
and CNS defects in mutant embryos (Mayer and NussleinVolhard, 1988; Klämbt et al., 1991).
We used the genomic sequences that cross-hybridized to the
putative Star transcript to isolate several cDNAs from an
embryonic Drosophila cDNA library (Zinn et al., 1988), all of
which showed cross hybridization at high stringency. The longest
cDNA, called K2C, is 4 kb in length, the same size as the transcript disrupted by the SF126 insertion. This cDNA contains a
poly(A) tract 18 nucleotides 3′ from a polyadenylation signal, and
primer extension analysis shows the 5′ end of this sequence to be
the site of transcription initiation (data not shown).
The genomic region encoding the K2C cDNA is large,
encompassing about 27 kb, and the extent of the controlling
region is unknown. To determine the ability of this cDNA to
rescue the Star phenotype, we made an hsp70 promoterconstruct containing the K2C cDNA. This construct was transformed into flies using P-element-mediated transformation
(Spradling, 1986). Two independent transformant lines, Starhs.8
and Starhs.7 were isolated. Both of these insertions map to the
second chromosome and are homozygous lethal. The
Starhs.7/Starhs.8 genotype is also lethal, suggesting that the
recessive lethality is due to the overexpression of the K2C cDNA
and not due to the sites of the insertions. A small number of
escapers can be recovered from both lines, and they have rough
eyes when raised without heat shock at room temperature (Fig.
4A), with 24% of the ommatidia showing extra R7-like cells.
To rescue the Star phenotype with the K2C cDNA, we used
the Starhs.8 insertion. In these experiments, the flies were not
heat shocked and the effects observed are probably due to the
influence of an unknown enhancer element on the hsp70
promoter. We first established that the transformant has a
single insertion of the hsp70-K2C construct mapping to the
47A band on the right arm of the second chromosome. Fifteen
independent recombinants were isolated, each with a copy of
S104E and the Shs.8 insertion on the same chromosome. All the
recombinants are homozygous viable and have wild-type eyes
(Fig. 4B,F). Since both Star− homozygotes and Starhs.8
homozygotes are lethal, this represents a reciprocal rescue, presumably because the loss of Star function in S−/S− balances out
the overexpression of Star protein in the Shs.8/Shs.8 genotype.
The rescue of Star embryonic lethality by the K2C cDNA
shows that K2C encodes a Star transcript.
The Shs.8 insert was also recombined onto a chromosome
containing the ast1 mutation. When homozygous, the ast1,
Shs.8/ast1, Shs.8 flies are viable and have normal eyes, as seen
both externally (Fig. 4D) and in sections (Fig. 4H). Thus, the
Shs.8 construct rescues the asteroid rough eye phenotype, as
well as the star lethality. These data are consistent with
asteroid representing a viable class of mutant alleles in the Star
gene and show that the K2C cDNA corresponds to this gene.
Sequence analysis of the Star cDNA
The K2C cDNA was sequenced in its entirety (see Materials
and Methods) and was found to contain a single large open
Fig. 2. Genetic interactions of Star and asteroid with EgfrElp. (A-F) SEMs of adult eyes; bar=66.7µm. (A) S1/+. The eye is moderately rough.
(B) Elp1/+. The eye is quite rough and slightly smaller than a wild-type eye (see Fig. 1A). (C) S1, +/+,Elp1. The eye is small and contains fewer
facets than in S1/+ or Elp1/+ eyes. (D) Df(2L)ast 2/+. The breakpoints of this deficiency, which eliminates both Star and asteroid function, are
21D1-2; 22B2-3. The eye is mildly rough. (E) Df(2L)ast2, +/+, Elp1. The eye is much smaller and rougher than Df(2L)ast2/+ or Elp1/+ alone
and has a more extreme phenotype than S1,+/+,Elp1 (compare with 2C). (F) Shs.8,+/Elp1. The eye is indistinguishable from Elp1/+ alone
(compare with 2B).
Star and its interactions with sevenless and EGF receptor in Drosophila 1737
reading frame (ORF), 1.8 kb in length, with approximately 1
kb each of both 5′ and 3′ untranslated sequences. Conceptual
translation of this ORF is shown in Fig. 3A. It encodes a
polypeptide of 598 amino acid residues, with a calculated
molecular mass of 66 kD. The Star ORF shows no sequence
similarity, with the exception of an opa repeat, a histidine-rich
region, and a glycine rich region, when compared with other
proteins in the PD2 data base using BLASTP (Altschul et al.,
1990). The ORF does not contain a signal sequence, however
it does contain a sequence starting at amino acid 280, that by
hydropathy analysis (Fig. 3A,D) is an excellent candidate for
a transmembrane spanning sequence. This sequence contains
22 hydrophobic residues, no internal proline residues, and is
flanked by charged amino acids. This putative transmembrane
sequence is located in the middle of the ORF and appears to
separate the protein into a highly hydrophilic amino-terminal
Fig. 3. Molecular analysis of Star. (A) Predicted amino acid sequence of the Star protein based on cDNA sequence analysis (nucleotide
sequence not shown but entered in data base). The putative transmembrane domain is underlined. Potential N-linked glycosylation sites are
noted with closed circles. The boundaries of the putative PEST sequences are marked by carets (residues 73-86 and residues 150-191). The
beginning of the 7 residue domain containing alternating positive and negative charges is marked with an open circle. (B) Northern analysis of
the Star transcript during embryonic development. Lanes 1: 0-6 hours; 2: 6-10 hours; 3: 10-14 hours; 4: 14-18 hours. Equal amounts of total
RNA were used in each lane to generate a developmental northern blot that was hybridized with a probe made from the Star cDNA, K2C.
(C) Genomic organization of the Star locus. The restriction map of genomic DNA that encodes the Star transcript and the site of the P-element
insertion causing the SF126 mutation are shown. The orientation of the P element is indicated by the direction of transcription of the βgalactosidase gene (shown by an arrow). R, EcoR1; S, Sal1; ry, rosy. (D) Hydropathy analysis of the predicted sequence of the Star protein.
Numbering of residues is the same as in A. Regions below the line indicate relative hydrophobicity and regions above the line relative
hydrophilicity. The putative transmembrane domain is marked with an arrow.
1738 A. Kolodkin and others
Fig. 4. Rescue of Star and asteroid eye phenotypes by the K2C cDNA. (A-D) SEMs (bar=66.7 µm) and (E-H) light microscope distal sections
(bar=1 µm), of adult eyes. (A,E) Shs.8/Shs.8. The Shs.8/Shs.8 genotype causes recessive lethality. A rare escaper is shown here that has a rough
eye. The section shows that most of the ommatidia are normal; in a small number of cases, there are extra R7-like cells. (B,F) S104E, dp,
Shs.8/S104E, dp, Shs.8. These flies are fully viable. The eye looks wild type both externally and in sections, showing a rescue of the roughness
associated with S104E and Shs.8. (C,G) ast1/ast1. The eye is reduced in size and very rough, but the ommatidia that do develop usually have the
normal complement of R cells. (D,H) ast1, dp, Shs.8/ast1, dp, Shs.8. The external morphology and ommatidial structure are completely wild type,
showing full rescue of the ast1 rough eye phenotype by the K2C cDNA.
domain of 279 amino acid residues, and a carboxy-terminal
domain of 296 residues that is relatively neutral with respect
to hydrophilicity (Fig. 3D). These characteristics are consistent
with the ORF encoding a type II membrane protein in which
the amino-terminal end is cytoplasmic and the residues
carboxy-terminal to the transmembrane domain are extracellularly located. Northern analysis utilizing equal amounts of total
RNA, free polysome-associated RNA and membrane-bound
polysome-associated RNA shows a large enrichment for the
Star transcript in RNA associated with membrane-bound
polysomes, suggesting that the Star transcript is localized in a
manner consistent with it encoding a membrane protein (data
not shown). There are five potential sites for N-linked glycosylation (Fig. 3A), two amino-terminal and three carboxyterminal to the transmembrane domain. In the putative aminoterminal cytoplasmic domain, there are two PEST sequences
thought to be associated with proteins that have short half lives
(Rogers et al., 1986). This cytoplasmic domain also contains a
stretch of alternating positive and negative charges (Fig. 3A).
This is similar to sequences in c-fos and N-myc leucine zippers
proposed to form an alpha-helix (Kohl et al., 1986; Landschultz et al., 1988). Alternating positively and negatively
charged amino acids are also found in the cytoplasmic domain
of the Toll product (Hashimoto et al., 1988) and the proposed
cytoplasmic domain of the Rhomboid protein (Bier et al.,
1990).
Expression of Star in the embryo and the eye disc
The embryonic expression of the Star transcript, as determined
by whole-mount in situ hybridization of digoxigenin (-DIG)labeled RNA probes, is temporally and spatially dynamic (Fig. 5).
We first detect expression in the early blastoderm (~stage 4;
staging as in Campos-Ortega and Hartenstein, 1985) in a longitudinal ventrolateral domain that is approximately 7-9 cells
wide (not shown). As gastrulation proceeds (stage 7) the transcript localization is seen to move ventrally and becomes
prominent in a row of cells approximately 5-7 cells wide on
either side of the ventral midline (Fig. 5A,B). During the initial
stages of germ-band elongation, the two lateral bands of the Star
transcript form a single band along the ventral midline that
initially is 6-10 cells in width but becomes narrower, so that by
stage 8 a single darkly stained band 2-4 cells wide can be seen,
with some reduced expression evident in the cells just lateral to
the midline. As germband elongation nears completion (stage
9-10; Fig. 5C,E), the midline expression becomes further
refined and is elevated in a single band of cells, 1-2 cells wide,
that corresponds to the mesectoderm. At this time, dorsoventral
epidermal stripes, faintly visible in early stage 7, are more
prominent and appear darkest in the central portion of each
segment. These stripes become more intense by stage 11-12
(Fig. 5D), and then decay so that by stage 15-16 they are not
visible. As germband retraction proceeds, the midline Star
expression is further refined to small clusters of cells (not
shown) that appear to be in the position of the midline glial cells
(Klämbt et al., 1991); however, certain identification of these
cells (and also lateral body wall cells in the position of PNS
organs) will require antibodies to the Star protein. Star
expression is also seen in cells of the embryonic brain in the
location of the optic lobe anlagen (Campos-Ortega and Hartenstein, 1985), starting at stage 12 and seen more clearly in stage
15 embryos (Fig. 5F). In addition to the tissues described above,
Star expression is seen at much lower levels in many other
Star and its interactions with sevenless and EGF receptor in Drosophila 1739
tissues between stages 4 and 15. This more general expression
is absent when a sense-strand riboprobe is used (not shown).
The results of hybridizing DIG-labeled RNA probes to late
third instar eye discs are shown in Fig. 6. High levels of the
Star transcript are expressed at the morphogenetic furrow in a
very narrow band that is only one cluster wide (Fig. 6A,B).
The expression is seen in clusters of cells that are regularly
spaced, with unstained cells in between. While these groups of
cells resemble the early developing photoreceptor clusters, the
resolution of this technique does not allow us to make assignments of this expression pattern to individual cell types. Immediately posterior to the morphogenetic furrow, the level of Star
transcript drops quite dramatically for three to four columns
before being expressed at high levels again in the developing
photoreceptor cell clusters (Fig. 6A,B). Initially the transcript
is strongly expressed in
three cells, which are likely
to be R2, R5 and R8 (Fig.
6D). Later in development,
other cells in addition to
R2, R5 and R8 express Star
(Fig. 6E); however, the resolution of this technique
does not allow us to
determine unambiguously
which cells in more mature
posterior
clusters
are
expressing the transcript.
Overexpressed Star
facilitates R7
development
To study further the role of
Star in R7 development,
we crossed a single copy
of the Starhs.8 insert into
the sensitized sevE4/Y;
SosJC2/+ background. As
shown in Fig. 7D and in
Table 4, this construct
dominantly enhances R7
development, raising the
level of suppression from
17% to 73%. In 13% of
these ommatidia an extra
R7-like cell is seen. We
have not yet established the
developmental origin of
these extra R7 cells. R8
cells develop normally in
this genetic background.
Moreover, Shs.8/CyO larval
eye discs stained with an
antibody against Boss,
exhibit no change in the
expression pattern of Boss
protein (not shown). Thus,
the enhancement of the
sevE4/Y;SosJC2/+ phenotype caused by overexpression of the Star gene is
unlikely to be a consequence of extra R8 cells or of boss misexpression. Since a small change in the level of Boss
expression will not be detectable in our assay, we cannot rule
out the possibility that the effect on R7 development is indirectly through controlling levels of Boss. The enhancement of
the sevE4/Y;SosJC2/+ eye phenotype and the suppression of
S1/S1 lethality by Shs.8 are effects seen without the application
of heat shock. We assume that this construct is under the
influence of an unknown enhancer element causing high levels
of expression at 25°C.
To express Star cDNA using a defined enhancer element,
we also isolated eleven independent transformants, sE-S1 to
sE-S11, in which Star is overexpressed in cells where Sevenless
is normally expressed using two sevenless enhancers fused to
the hsp70 promoter. In contrast to the hs.8 transformant
Fig. 5. Embryonic expression of the Star transcript. Whole-mount in situ analysis using DIG-labeled Star
cDNA probes. Anterior is to the left. (A,B) Stage 7 (ventral view; staging as in Campos-Ortega and
Hartenstein, 1985) embryo showing Star transcript localization restricted to a row of cells approximately 5-7
cells wide on either side of the ventral midline (arrowheads). (C,E) Stage 9-10 embryo (ventral view)
showing midline Star expression restricted to a single row of cells, 1-2 cells wide (arrows), that corresponds
to the mesectoderm. Dorsoventral epidermal stripes, visible on either side of the midline (arrowheads),
appear restricted to the central portion of each segment. (D) Stage 11 embryo, lateral view, showing
segmentally repeated epidermal stripes (arrowhead). (F) Star expression in the optic lobe anlagen of the
embryonic brain (arrowhead) as seen in a lateral view of a stage 15 embryo. Bar: (A-D),F: 50µm; E: 25µm.
1740 A. Kolodkin and others
described above, all of the sE-S lines are homozygous viable,
consistent with the expression being controlled by the eyespecific sevenless enhancer (sE). Ten of the lines, sE-S1 to sES10, show a rough eye when two copies of the insertion are
present. For sE-S1 to sE-S9 a single copy of the insertion gives
rise to an eye with wild-type appearance, both externally and
in sections. For sE-S10, the eye is mildly rough when one copy
of the insertion is present. Since the phenotypes for these lines
are qualitatively similar to one another, they were analyzed
further. The eleventh line, sE-S11, shows no roughness, either
in one or two copies. Since the phenotype of this atypical line
may be influenced by position effects or is due to its site of
insertion, it is not described further in this paper.
The external eye morphology due to one of the ten typical
insertions is shown in Fig. 8A and B. The roughness of these
eyes is likely to be due to defects in cone and pigment cells.
The defects in photoreceptor cell development can be seen in
sections. Qualitatively the phenotypes are similar in all lines,
but quantitatively they are quite variable (compare Fig. 8C
with D). In the weakest line, sE-S3, 99% (n=460) of the
ommatidia are wild type and 1% of the ommatidia show an
extra cell with either outer cell or R7-like morphology (Fig.
8C). In the strongest insertion, sE-S10, 26% of the ommatidia
contain extra cells with R7-like morphology, while 27% of the
ommatidia have extra cells with R1-6 morphology (n=273).
In order to determine the effects of the sE-S insertions on
R7 development, these lines
were crossed into the sensitized
sevE4/Y; SosJC2/+ background.
All six lines tested act as
enhancers of R7 cell development (see Table 5). While we
cannot rule out that some of this
enhancement is due to leaky
expression from the hsp70
promoter in R8 cells, the fact
that all these sE-S lines act as
enhancers suggests to us that
expression due to the sevenless
enhancer as lead to the development of R7 cells. These results
will be further confirmed using
a sevenless promoter construct
now available (Fortini et al.,
1993).
Star function is necessary
for early ommatidial
assembly
For most essential genes, a later
function in photoreceptor cell
development can be assessed by
analyzing mosaic patches of
mutant tissue in the adult eye.
This is not possible for Star,
since homozygous patches
scored in the adult eye are seen
as scars containing no photoreceptor cells (Heberlein and
Rubin, 1988). This is a terminal
phenotype which could have any
number of primary causes. To identify the primary cause for
this defect, we made homozygous mutant patches of the strong
S1 allele in the larval eye disc. We used the Flp recombinase
method (Golic, 1991) to generate homozygous mutant clones at
high frequencies. The eye discs were stained using the neuronspecific antibody mAb22C10 and 60 independent homozygous
clones, with no staining were identified. Patches were generated
at an estimated frequency of 80% (see Materials and Methods
for details). As shown in Fig. 9, the phenotype of these clones
is a complete absence of staining with mAb22C10. The mosaic
clones were also double stained with mAb22C10 and an
antibody against Boss which is specific to the R8 cell. As
expected, the mutant clones do not stain with the Boss antibody
either (not shown). Defects of this nature are never seen in wildtype controls stained with mAb22C10 or Boss antibody. This
result suggests that neuronal development does not take place
in the eye disc if Star gene function is missing. Similar observations have been made by Heberlein et al. (1993) who have
further shown evidence for cell death when Star function is
lacking.
DISCUSSION
In this paper, we report the cloning of the Star gene by Pelement tagging. Excision of the P element gives rise to both
Fig. 6. Expression of the Star transcript in third instar larval eye discs. Whole-mount in situ analysis
on eye discs; posterior to the left. (A,B) Two different examples of eye discs stained in situ with the
Star K2C riboprobe are shown. In both cases, a narrow band of signal is seen at the morphogenetic
furrow (arrows). The staining is in discrete clusters spaced at regular intervals. The expression level
drops sharply posterior to the furrow and rises again 3-4 columns later in developing clusters. (C) No
signal is seen in control discs hybridized to a sense RNA probe. (D) The second wave of Star
expression is restricted to three cells in each developing cluster, corresponding to the positions of
R2, R5 and R8 in the developing clusters. The disc shown in this panel is young, and only one
column of clusters shows Star expression. (E) In an older disc, several columns of clusters show Star
expression in three cells only (empty arrow). In more mature clusters, more than three cells (solid
arrow) express Star. The resolution of this technique does not allow us to unambiguously identify
the cells in the more mature clusters.
Star and its interactions with sevenless and EGF receptor in Drosophila 1741
Star and asteroid mutants. A 4 kb cDNA from the region of
the insertion rescues the embryonic lethality of Star mutants
and the eye phenotype of asteroid mutants. Taken together, the
rescue and excision results imply that asteroid and Star are
different classes of mutations in the same gene. The Star gene
encodes a 4 kb transcript, which is expressed at the morphogenetic furrow and in the posterior developing clusters in third
instar eye discs. In embryos, this transcript is expressed in
several tissues, most notably in the mesectoderm and some of
its derivatives. The conceptual translation product of the gene
suggests that it could encode a novel type II membrane protein,
with a hydrophilic intracellular domain and an extracellular
domain that is neutral with respect to hydrophilicity. Internal
hydrophobic domains are known to mediate membrane
insertion of type II membrane proteins, resulting in the
hydrophilic amino terminus facing the cytoplasm and an exoplasmic carboxy terminus (Spiess and Lodish, 1986). The
charge distribution in the amino terminus of the Star protein is
consistent with this interpretation (reviewed in Dalbey, 1990).
The amino terminus, particularly the residues adjacent to the
putative transmembrane domain, are highly positively charged.
The function of Star is fairly widespread during Drosophila
Table 4. Shs.7 and Shs.8 enhance SosJC2
Genotype
sevE4/Y; SosJC2/+
sevE4/Y; SosJC2/+, Shs.8
sevE4/Y; SosJC2/+, Shs.7
Ommatidia with
central cells (%)
Total ommatidia
counted
16
73
65
2392
2000
2185
development. Detailed analysis of Star function in embryonic
CNS development has established its importance in the correct
differentiation of midline glia (Klämbt et al.,1991). Star also
functions in germ line, embryonic PNS and establishment of
embryonic dorsoventral patterning (Mayer and NussleinVolhard, 1988; Rutledge et al., 1992; Bier et al., 1990). A
genetically sensitized screening technique, involving a
dominant allele of the Son of sevenless gene, allowed us to
characterize a role for Star in photoreceptor cell development.
This technique utilizes the appearance of R7 cells in a genetic
background where the components of the sevenless signaling
system are present in limiting amounts. A twofold change in
the dose of many gene products involved in R7 development
can be detected. A particularly useful feature of this assay is
that it can identify genes specific to the development of R7 (e.g.
boss), as well as genes whose function is not limited to the
development of R7 (e.g. Ras1). Thus, molecules with widespread roles in development can be isolated, and their particular role in R7 development can be determined. A unique feature
of this screen is the fact that components upstream and downstream of Sos can be detected. Furthermore, this assay can
detect mutations in a single copy of these genes and therefore
is able to identify recessive lethal mutations. Since the
Sevenless and EGF receptors have been placed in genetically
defined pathways that share several downstream molecules
(Simon et al., 1991; Bonfini et al., 1992), it is not surprising
that many of the genes identified in this screen participate in
both pathways.
All of the above features were important for us to be able to
isolate alleles of Star in this screen. Mutations in Star are
embryonic lethal, implying Star functions early in development. In the eye, mosaic analysis showed that Star function is
genetically required in R2, R5 and R8 (Heberlein and Rubin,
1991), suggesting that its role in R7 development is likely to
be non-autonomous and upstream of the Sevenless receptor.
Table 5. sevenless enhancer-Star cDNA lines enhance SosJC2
Genotype
Fig. 7. Shs.8 promotes R7 development. (Bar=1 µm). (A) Shs.8/+. In
heterozygotes, containing only one copy of the K2C cDNA insert, all
ommatidia are wild type. (B) Shs.8/Shs.8. In escapers of this genotype,
24% of the ommatidia contain extra R7-like cells. (C) sevE4;
SosJC2/+. In this genetic background, 16% of the ommatidia contain
a R7 cell. (D) sevE4; SosJC2/+, Shs.8. In the sensitized genetic
background, 73% of the ommatidia develop R7 cells. This is a
significant increase over the percentage of R7 cells seen in C (see
Table 4).
sevE4/Y; SosJC2/+
sevE4/Y; SosJC2/+, sE-S3
sevE4/Y; SosJC2/+, sE-S4
sevE4/Y; SosJC2/+, sE-S5
sevE4/Y; SosJC2/+; sE-S6/+
sevE4/Y; SosJC2/+; sE-S7/+
sevE4/Y; SosJC2/+; sE-S10/+
Ommatidia with
central cells (%)
Total ommatidia
counted
16
60
28
44
35
56
91
2392
3575
3938
4144
2159
3753
262
The levels of suppression are determined by the pseudopupil method,
counting at least 2000 ommatidia for each line as in Table 1, except for the
sE-S10 transformant. This line gives dominant rough eyes which makes
counting by pseudopupil inaccurate. The number of R7 cells in the eyes of
sevE4/Y; SosJC2/+; sE-S10/+ flies was determined from sections of adult eyes.
1742 A. Kolodkin and others
However, other possibilities are discussed later. Star also
interacts with the EGF receptor, both in the eye and in other
tissues, suggesting that it plays a more general role in allowing
cells to interact with each other in different contexts. We have
found, using mosaic analysis in developing eye discs, that loss
of Star function at the furrow prevents R cells from developing. This implies that Star is required at an early step in the
establishment of the identity of R8, R2 and R5.
A construct containing the Star cDNA under the control of
the heat-shock promoter rescues the embryonic lethality
caused by Star mutations. Embryonic defects seen in Star
mutants include deletion of structures along the ventral midline
resulting in loss of medial portions of the denticle belts,
deletions of certain components of the PNS, and CNS midline
defects. Though our analysis of Star expression is limited by
the resolution of the whole-mount in situ technique, the pattern
of expression that we observe is consistent with the phenotypes
seen in Star mutations. The Star transcript is first seen in longitudinal lateral stripes that move ventrally during early
embryonic development and resolve, by stage 9-10, into a
single stripe 1-2 cells wide along the ventral midline. The Star
CNS defect results in fusion of the two commissures, and
analysis of the midline glia that appear to be essential for
proper separation of the commissural tracts reveals that, in Star
mutant embryos, these glia do develop, but subsequently fail
to migrate and die (Klämbt et al., 1991). Star is expressed in
the mesectoderm, which gives rise to the midline glia, and later
during development in cells along the
midline that are likely to be the midline glia.
The additional early expression of Star in
cells lateral to the mesectoderm and later
expression in epidermal stripes, in addition to
weak expression in the region of the lateral
PNS organs, is also expected given the
embryonic phenotypes associated with Star
mutations (Kim and Crews, 1993); however,
antibodies to the Star protein will be needed
to characterize more fully these aspects of
Star expression.
Star and rhomboid share common
features
Within the spitz group genes, only rhomboid
and Star have so far been implicated in eye
development. These two genes also seem to
have similar functions in other tissues. In the
embryonic CNS, both rhomboid and Star
affect the development of the midline glial
cells (Klämbt et al., 1991). In the embryonic
PNS, both mutants have similar reductions in
the number of specific sensory organs
(Mayer and Nusslein-Volhard, 1988; Bier et
al., 1990). Both Star and rhomboid function
in wing development, since Star strongly
enhances the wing phenotype of the rhoVE
allele. Based on this result and other genetic
interactions, Sturtevant et al. (1993) have
proposed that Star and Rhomboid may
interact directly in the wing imaginal disc to
enhance a ventrolateral signal which controls
longitudinal wing vein development. Also,
the sequences of Star and Rhomboid share certain characteristics. Both are putative integral membrane proteins with no
signal sequence, contain highly charged amino-terminal
segments and contain PEST sequences. Additionally, both Star
and rho are expressed in overlapping embryonic tissues, most
significantly in the mesectoderm and in similar patterns in the
third instar larval eye disc. In the eye, the Rhomboid protein
is strongly expressed in R2, R5 and R8 (Freeman et al., 1992),
the same cells in which Star function is required for correct
ommatidial assembly and in which high levels of Star
expression are seen. When we tested strong rhomboid
mutations in the sensitized sevE4; SosJC2 background, there was
no significant effect on the level of suppression (data not
shown). However, since rhomboid may have a redundant role
in photoreceptor development (Freeman et al., 1992), this does
not rule out a function for rho in R7 determination. Overexpression of either Star or rhomboid can give rise to supernumerary photoreceptor cells with outer cell morphology (Fig.
8C,D; Freeman et al., 1992). While the expression pattern in
the eye disc and the phenotypes of the overexpressed gene
products suggests that Star and Rhomboid may function in
common processes in eye development, it remains to be seen
whether they are members of the same pathway.
An early and a late role for Star in eye development
Based on our genetic analysis, we propose that Star function
is necessary for the determination of R cell fate, both early at
Fig. 8. Star cDNA expressed under the control of the sevenless enhancer promotes
supernumerary R cell development. (A,C) SEMs of adult eyes; bar=66.7 µm.
(B,D) Light microscope sections of adult eyes; bar=1 µm. (A) A single copy of sE-S3
leads to a wild-type eye. (C) In two copies, sE-S3/sE-S3 gives a mild rough eye. (B) sES4/sE-S4. This is an example of a transformant with a mild phenotype. This section, at
the R7 plane, shows one example of an ommatidium with an extra outer cell (arrow).
The position of this extra cell resembles the position of the extra outer cells seen when
rhomboid is expressed using the sevenless enhancer (Freeman et al. 1992). It is likely
that this cell results from the transformation of a mystery cell into a R cell. (D) sES3/sE-S3. An example of an insertion with a strong eye phenotype. Sections taken at the
R7 plane show ommatidia with extra outer cells (arrow) as well as those with extra R7like cells (arrowheads).
Star and its interactions with sevenless and EGF receptor in Drosophila 1743
Fig. 9. Mosaic analysis of Flp-induced S1/S1 patches in the larval eye disc. All eye discs were stained with the neuron-specific marker mAb
22C10. (A,B) Two different examples of clones homozygous for the strong S1 allele. The arrowheads delimit the boundaries of the clones. The
clones do not stain with mAb22C10, indicating that cells of a S1/S1 genotype fail to develop as photoreceptors. (C) The same clone as in B, at a
higher magnification, showing that there are single cells that stain with mAb22C10 along the mosaic boundary, but cells within the patch are
unstained.
the morphogenetic furrow and also later, when the R7 cell
identity is established. The early expression of Star in R8, R2
and R5 at the furrow and the later expression in more cells in
the developing clusters is consistent with this notion.
Mosaic analysis in the adult eye demonstrates that Star is
required in the first three photoreceptor cells to develop
(Heberlein and Rubin, 1991). The early role of Star in R cell
development was further substantiated by generating mosaic
patches in the larval eye disc. We found that in the absence of
Star function, R2, R5 and R8 fail to develop and, perhaps as a
consequence, the rest of the R cells also do not develop.
It could be argued that Star has no role to play in the development of R7, instead its only role is in the earlier step when
R8 identity is established. In this model, Star ceases to function
once R8 development is complete and any effects on R7 development are simply a consequence of the R8 cell not developing at all. Our results do not support such a view. While it is
certainly true that mosaic analysis suggests that complete loss
of Star function will prevent R8 cells from developing, other
genetic tests clearly show that in a wild-type fly, the function
of Star is needed at a later stage when R7 cells develop. One
line of evidence for this is the effect of increased levels of Star
on the development of R7 cells in the sensitized genetic background provided by the sevE4; SosJC2 double mutant. In this
background, the R8 cell is normal, but the sevenless-mediated
developmental signal is attenuated. When the level of Star is
increased, the number of ommatidia with an R7 cell goes up
dramatically. This process is dependent on Sos and sevenless,
and is therefore utilizing the normal induction process that
leads to the development of the R7 cell. Furthermore, while
Star−/+ and Sos−/+ flies have negligible defects in R7 development, a synergistic effect is seen in S−, +/+, Sos− ommatidia,
which often fail to develop R7, even though an R8 cell
develops in every ommatidium. Taken together, the results
from genetic backgrounds involving gain or loss of function of
Star lead us to conclude that an alteration in the level of Star
protein not only affects the early determination of R8, but also
subsequently affects the proper development of the R7 cell.
Mosaic analysis in adult eyes has suggested a role for Star
in R2, R5 and R8 for proper ommatidial assembly (Heberlein
and Rubin, 1991). The simplest conclusion from the mosaic
analysis is that Star function is required non-autonomously in
the R8 cell for the R7 cell to develop. The results from this
study suggest that the role of Star may be more complex. While
early developing clusters express Star in R2, R5 and R8, in
mature photoreceptor cell clusters, Star is expressed in other
R cells. Also, overexpression of Star in the sE-Star constructs
promotes R7 development, although the sevenless enhancer
does not give expression in R8, R2 and R5. Two hypotheses
can be proposed. Star may have a redundant, but autonomous
function in R7, not evident in adult mosaic patches, and only
becoming apparent in a sensitized genetic background where
the R7 developmental signal is attenuated. Alternatively, Star
function could be required in both R7 and R8, but loss of Star
function from either one of these cells is not detrimental to the
development of R7. Thus, in the mosaic patches, Star− R7 cells
can develop if R8 is Star+. Since the early role of Star in R8
cells is essential for neuronal development, an ommatidium
with a mutant R8 would never initiate a normal cluster.
Star functions in two different signal transduction
pathways
We have found that Star interacts genetically with mutations
in EGFR and Sevenless, both of which function in the eye. We
hypothesize that the early role of Star is important for R8
development and is mediated by the participation of Star in the
EGF receptor pathway and that the late role of Star, important
for R7 induction, is mediated by an involvement in the
Sevenless pathway. Star is also involved in many aspects of
embryonic development that require the EGFR, including the
development of structures that derive from the ventral midline.
A likely function for Star could be enhancing or amplifying
signals exchanged by different signal transduction systems,
thereby enabling a cell to acquire its differentiated state. Future
biochemical analysis will reveal if this model is indeed correct.
We are grateful to Ulrike Heberlein and Gerald Rubin, and to Ethan
Bier for communicating unpublished results. We thank Yasushi
Hiromi for the P insertion line SF126, Eric Schwartz for Star and
asteroid stocks, Barry Dickson and Ernst Hafen for the sevenless
enhancer construct, Lily Jan and Yuh N. Jan for the FRT and Flp
1744 A. Kolodkin and others
stocks, and Kathy Matthews for sending us many Drosophila lines.
We are indebted to Ronald Rogge for photographic and other assistance. We thank David VanVactor, Larry Zipursky, Steve Crews and
members of the Banerjee laboratory for comments on the manuscript.
This work was supported by a Damon Runyon postdoctoral fellowship to A. L. K., a National Institutes of Health predoctoral
traineeship to D. L. and by a McKnight Scholars' award, an Alfred P.
Sloan Foundation fellowship, and a National Institutes of Health grant
to U. B. (FDP USHHS 1 R01 EY08152-01A1). C. S. G. is an Investigator with the Howard Hughes Medical Institute.
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(Accepted 30 March 1994)
Note added in proof
GenBank Accession number is L31886.