Cell, Vol. 96, 847±856, March 19, 1999, Copyright 1999 by Cell Press
The Transmembrane Molecule Kekkon 1 Acts in a
Feedback Loop to Negatively Regulate the Activity
of the Drosophila EGF Receptor during Oogenesis
roles, since quantitative variations intheirlevelofactiva-
tions can lead to very different cellular outcomes. For
example, studies in mammalian PC12 cells have illus-
trated that prolonged MAPK activation and nuclear
translocation triggers differentiation, whereas transient
activation leads to proliferation (see Li and Perrimon,
1997). Similarly, in the Drosophila embryos, patterning
of the terminal region is determined precisely by the
strength of the signal generated by the Torso RTK
(Greenwood and Struhl, 1997). Thus, precise regulation
of the quantitative output of a signal generated by an
RTK is critical to the control of growth and differenti-
While many positive regulators/effectors of RTK sig-
naling activity have beencharacterized, only a few inhib-
itory molecules have been identified (Freeman et al.,
1992; Kharitonenkov et al., 1997). In addition to these
inhibitory molecules,a numberofinhibitory mechanisms
have been identified. These include internalization of
ligand±receptorcomplexes, receptordesensitization as
a result of hyperphosphorylation of the receptor, in-
hibitory regulation by cytoplasmic and transmembrane
protein phosphatases, and targeting to the proteolytic
pathway of specific signal transducers (Ullrich and
Schlessinger, 1990; Sturtevant et al., 1994; van derGeer
et al., 1994; Kokel et al., 1998). By integrating particular
stimulatory and inhibitory mechanisms in cells, RTK ac-
tivities can be quantitatively regulated to direct distinct
The Drosophila EGFR is required for a multitude of
developmental processes throughout the life cycle (re-
views by Ray and Schupbach, 1996; Perrimon and Per-
kins, 1997; Schweitzer and Shilo, 1997). For example,
activation of the EGFR pathway in follicle cells during
oogenesis establishes both anteroposterior and dorso-
ventralfates. During embryogenesis, the EGFR is required
forthe establishment of ventral cell fates, determination
of segmental identity, maintenance of amnioserosa and
ventralneuroectodermalcells, germband retraction, cell
fate specification in the central nervous system, and
production of cuticle. During larval development, the
EGFR plays a role in cell proliferation, vein formation,
and eye development. These multiple tissue-specific ac-
tivities are regulated in part by different ligands that con-
tainanEGF repeat similarto that oftransforming growth
factor ? (TGF?), a known ligand of the vertebrate EGFR.
To date, three such ligands have been isolated in Dro-
sophila: Spitz (Spi; Rutledge et al., 1992; Schweitzer et
al., 1995a), Gurken(Grk;Neuman-Silberberg and Schup-
bach, 1993), and Vein (Vn; Schnepp et al., 1996). In con-
trast to these stimulatory molecules, Argos (Aos), a se-
creted protein with an atypical EGF motif, has been
showntoactas anantagonistofEGFR activity(Schweitzer
et al., 1995b). In vitro studies have revealed that Aos
can interfere with EGFR activation in a manner that is
both saturable and competitive, and it has been pro-
posed thatAos acts as a directinhibitorofSpiby binding
directly to the EGFR (Schweitzeret al., 1995b). Remark-
ably, the expression of aos is dependent upon EGFR
activation (Golembo et al., 1996b), thus demonstrating
Christian Ghiglione1, Kermit L. Carraway III3,
Laufey T. Amundadottir1,6, Robert E. Boswell4,
Norbert Perrimon1,2,5, and J oseph B. Duffy1,4,5,7
1Department of Genetics
2Howard Hughes Medical Institute
Harvard Medical School
Boston, Massachusetts 02115
3Division of Signal Transduction
Beth Israel Deaconess Medical Center
Boston, Massachusetts 02115
4Department of Molecular, Cellular,
and Developmental Biology
Howard Hughes Medical Institute
University of Colorado
Boulder, Colorado 80309
We have identified the Drosophila transmembrane
molecule kekkon 1 (kek1) as an inhibitor of the epider-
mal growth factor receptor (EGFR) and demonstrate
that it acts in a negative feedback loop to modulate the
activity ofthe EGFR tyrosine kinase.During oogenesis,
kek1 is expressed in response to the Gurken/EGFR
signaling pathway, and loss of kek1 activity is associ-
ated with an increase in EGFR signaling. Consistent
withourloss-of-functionstudies, we demonstrate that
ectopic overexpression of kek1 mimics a loss of EGFR
activity.We show that the extracellularand transmem-
brane domains ofKek1caninhibitand physically asso-
ciate with the EGFR, suggesting potential models for
this inhibitory mechanism.
The biological processes regulated by receptortyrosine
kinase (RTK)signaling pathways are diverse and include
the regulation of cell growth, differentiation, migration,
viability, and maintenance of homeostasis (see review
by Ullrichand Schlessinger, 1990). Inthe pastfew years,
a great deal has been learned about the mechanisms
by which RTKs transduce signals and specify particular
cellularfates. Inparticular, mostRTKs have beenshown
to regulate gene expression through the Ras/Raf/MEK/
MAPK cassette (see Li and Perrimon, 1997). Although
the pathways that transduce signals from RTKs to the
nucleus have been well characterized, less is known
about the mechanisms involved in imparting specificity.
More recently, however, it has beenshownthat modula-
tionofRTK activities is acriticalaspectoftheirbiological
5To whomcorrespondence should be addressed (e-mail:perrimon@
rascal.med.harvard.edu [N. P.], email@example.com [J . B. D.]).
6Present address: deCode Genetics, Lynghals 1, 110 Reykjavik,
7Presentaddress:DepartmentofBiology,Indiana University,J ordan
Hall, Bloomington, Indiana 47405.
that the EGFR regulates the expression of its own nega-
During lateoogenesis,EGFR activityis regulatedby all
ofthesemechanisms, and its precise activity is criticalin
specifying the properpatternof the chorionand embryo
(review by Ray and Schupbach, 1996). Thus, function
of the EGFR during oogenesis provides an excellent
paradigm to characterize the quantitative regulation of
the EGFR and determine if additional components of
this pathway exist. In response to Grk signaling from
the oocyte, activationofthe EGFR infollicle cells follows
a dynamic pattern. In early egg chambers, Grk first acti-
vates the EGFR pathway in posterior follicle cells. Sub-
sequently, around stage 7, grk transcripts become local-
ized to the anterodorsal corner of the oocyte and signal
to adjacent follicle cells to define theirdorsalfates. Acti-
vated EGFR molecules regulate transcription through
the Ras/Raf/MEK/MAPK cassette in follicle cells (Brand
and Perrimon, 1994; Hsu and Perrimon, 1994; Schnorr
and Berg, 1996). In a screen to identify downstream
genes regulated by the EGFR in follicle cells, we have
identified the gene kekkon 1 (kek1), which encodes a
single pass transmembrane protein with features of a
cell adhesion molecule with leucine-rich repeats (LRR)
and immunoglobulin (Ig) motifs (Musacchio and Perri-
mon, 1996), as a target gene. We demonstrate that en-
dogenous kek1acts inaninhibitory manner: loss ofkek1
activity results in increased EGFR signaling. We also
demonstrate that overexpression of kek1 blocks the ac-
tivity of the EGFR. This inhibition involves a physical
association between the extracellular and transmem-
brane domains of Kek1 with the EGFR. This establishes
Kek1 as a bona fide inhibitor of the EGFR that acts in
a negative feedback loop to modulate the activity of this
RTK signaling pathway.
Figure 1. kek1 Is a Transcriptional Target of the EGFR Pathway
(A±C)Expressionof kek1 inthe follicle cells revealed by the kek-lacZ
enhancertrap insertion. kek-lacZ expressioncanfirst be detected in
region 2 of the germarium (A). Expression continues to be detected
at the poles of stage 1±6 egg chambers. Following the dorsal±
anteriormigrationofthe oocyte nucleus, kek-lacZ expressionunder-
goes a similarshiftto a dorsal±anteriorregionofthe follicularepithe-
lium in stage 8 and 9 egg chambers (B). The position of the oocyte
nucleus is marked by the arrow. A lateral view of a stage 10 egg
chamber showing the dorsal±anterior graded expression of kek-
(D±F)kek-lacZ expressionis underthe controlofthe EGFR signaling
pathway. In females homozygous for the hypomorphic Draf muta-
tion, DrafHM7(Melnick et al., 1993) kek-lacZ expression is severely
reduced (D). Expression of kek-lacZ is completely abolished in ova-
ries of females homozygous for grkHK36(E) or mutations in the egfr
associated with female sterility (data not shown). Ectopic activation
of the EGFR receptor pathway using the gain-of-function Draf gene
hsDrafgof-F22(F) leads to uniform kek-lacZ expression. Induction of
WT Draf during oogenesis had no effect on the expression of kek-
lacZ (not shown).
(G and H) The position of the oocyte nucleus defines the spatial
domain of kek1 expression. In response to colchicine treatment, the
oocyte nucleus and its associated grk mRNAs become misplaced
during oogenesis (G and H). In these egg chambers, the expression
of kek-lacZ can be detected posteriorly as well as ventrally. In (G)
and (H), the germline nuclei are marked with the enhancer trap line
es(3)79 to allow easy detection of the oocyte nucleus (indicated by
(C±H) are stage 10 egg chambers oriented with dorsal up. Anterior
is to the left in all panels.
kek1 Is Regulated by the Grk/EGFR Pathway
in Follicle Cells
Tocharacterizetargetgenes oftheEGFR signaling path-
way, we screened a collection of enhancertrap lines for
expression patterns in follicle cells. We found one line,
15A6, which was expressed in a pattern consistent with
it being a regulatory target of the EGFR pathway, since
it undergoes a transition during stages 8±10 when it
relocalizes froma posteriorto a dorsal±anteriorgradient
(Figures 1A±1C). 15A6 corresponds to an insertion in
the kek1 gene (Musacchio and Perrimon, 1996), and we
refer to it as kek-lacZ in the text. We found that kek-
lacZ expression reflects accurately the expression of
kek1 both in wild-type (WT) and in mutant backgrounds
(data not shown).
To establish a regulatory link between the transcrip-
tional regulation of kek1 in the follicle cell epithelium
and the Grk/EGFR signaling pathway, we examined kek-
lacZ expression in mutants for grk, egfr, and the kinase
Draf that is both necessary and sufficient for EGFR sig-
naling in follicle cells (Brand and Perrimon, 1994). In
each case, the dorsal±anterior gradient of kek-lacZ ex-
pression was abolished or severely diminished (Figures
1D and 1E; see also Brand and Perrimon, 1994;Queenan
et al., 1997; Sapir et al., 1998), revealing that kek-lacZ
expression is transcriptionally regulated by the Grk/
EGFR/Draf pathway. Expression of an activated form of
Draf resulted in the ubiquitous expression of kek-lacZ
within the follicular epithelium (Figure 1F).
To further analyze the role of the Grk/EGFR pathway
in the transcriptional control of kek-lacZ, we mislocal-
ized the subcellular localization of grk mRNAs by dis-
rupting the oocyte cytoskeletal network (Figure 1G).
A Novel Regulator of the Drosophila EGF Receptor
Figure 2. Overexpression of kek1 during Oo-
genesis Blocks the Activity of the EGFR
Phenotypes of eggs and embryos derived
from WT (A), T155; UAS-kek1 (B), and T155;
UAS-egfrDN (C) females. The eggshell is se-
creted by the follicle cells during oogenesis.
Specialized groups offollicle cells onthe dor-
sal side of the egg chamber form the two
dorsal respiratory appendages. Note that the
chorionic filaments, localized anteriorly in the
WT, are fused, do not elongate, and are posi-
tioned more posteriorly when either kek1 or
the egfrDN is expressed uniformly in follicle
cells using the Gal4 driver T155. The embry-
onic cuticles that develop from these eggs
are strongly ventralized (cf. Figures B2 and
C2 with the WT in Figure A2), as observed in
embryos derivedfrom grk
mothers (Roth and Schupbach, 1994). This
ventralization phenotype can be readily de-
tected using the Twi marker for ventral cell
fates. In WT embryos (A3 and A4), Twi is ex-
pressed in the ventral-most region of the em-
bryo. The Twi expression domain is enlarged
when either kek1 or the egfrDN is misex-
pressed (B3, B4, C3, C4).
In all panels, anterior is to the left. The chorions shown in (A1), (B1), and (C1) are dorsal views. The embryos in (A2), (B2), (C2), (A3), (B3), (C3),
and (A4), (B4), and (C4) are lateral and ventral views, respectively.
Treatment of the oocyte with colchicine, an inhibitor of
microtubule polymerization, leads to mislocalization of
theoocytenucleus and its associated grkmRNAs, which
correlated with ectopic kek-lacZ expression (Figure 1H)
as well as kek1 mRNA expression (not shown). Alto-
gether, these results demonstrate that Grk is not only
necessary but sufficient to regulate the spatial expres-
sion of kek1.
Inadditionto its role inestablishing the dorsalcharac-
teristics ofthe eggshell, the Grk/EGFR pathway controls
embryonic dorsoventral patterning by restricting a ven-
tralizing signal (Neuman-Silberberg and Schupbach,
1994; Morisato and Anderson, 1995). As a result, loss of
EGFR functionleads to ventralized embryos. Embryonic
cuticles derived from T155; UAS-kek1 females showed
a ventralized cuticle phenotype (Figure 2B2). To analyze
the extent of this ventralization, we stained embryos for
Twist (Twi) RNA and protein that label a ventral domain
ten cells wide (Thisse et al., 1988). Overexpression of
kek1 in follicle cells results in an expansion of the Twi
expressiondomain(Figures 2B3and 2B4).Similarly, em-
bryos derived from T155; UAS-egfrDN females are also
strongly ventralized (Figures 2C2±2C4).
Altogether, our results indicate that overexpression
of kek1 in follicle cells ventralizes both the eggshell and
the embryo by most likely interfering with the activity of
the Grk/EGFR signaling pathway.
Overexpression of kek1 during Oogenesis
Antagonizes EGFR Activity
Previous analysis has revealed that in the complete ab-
sence of kek1 gene activity, flies are viable, fertile, and
do not exhibit any overt morphological defects (Musac-
chio and Perrimon, 1996). Kek1 has the features of cell
adhesionmolecules (CAMs), and loss-of-functionmuta-
tions of many CAMs have subtle mutant phenotypes.
However, when ectopically overexpressed, some CAM
molecules cangenerate striking mutantphenotypes that
are revealing of their functions (Speicher et al., 1998).
Thus, to gain insights into the possible function of kek1
during oogenesis, we tested the effect of overexpress-
ing kek1 in the follicle cells using the GAL4/UAS system
(Brand and Perrimon, 1993).
Whenkek1 is expressed underthe controlofthe GAL4
line T155, which drives expression all over the follicle
cellepitheliumbeginning atstage9(Harrisonetal., 1995;
Queenan et al., 1997), 100% of the resulting eggs are
longer than the WT eggs and have a reduction or a
complete loss ofdorsalappendages (cf. Figure 2B1 with
the WT in Figure 2A1). This loss of dorsal appendage
material is due to a ventralization of the eggshell, a
phenotype associated with egfr, or grk loss-of-function
mutations (Schupbach, 1987;Priceetal., 1989).A similar
ventralized eggshell phenotype was obtained when a
dominant-negative form of the EGFR, EGFRDN, was
expressed under the control of T155 (Figure 2C1).
Kek1 Inhibits the Activity of
the Ras/Raf/MEK/MAPK Cassette
To determine whetherKek1 blocks the signaling activity
of the EGFR, we tested whether ectopic expression of
regulated by the EGFR/Ras/Raf/MEK/MAPK pathway in
follicle cells. Since kek1 is a target of this pathway, we
used kek-lacZ as a reporterforthis experiment. Overex-
pression of kek1 using the Gal4 driverT155 was associ-
ated with a strong but not complete reduction of kek-
lacZ expression (cf. Figure 3B to WT inFigure 3A). Using
a stronger Gal4 line, CY2 (Queenan et al., 1997), a com-
plete disappearance of kek-lacZ expression was ob-
served (Figure 3C). These data are consistent with the
model that Kek1 downregulates the activity of the Ras/
Raf/MEK/MAPK pathway.Consistentwiththis, epistasis
experiments indicate that the inhibitory effect of Kek1
ing that Kek1 can block the effect of Rho on EGFR
activation. Altogether, these results place Kek1 up-
stream of the EGFR and downstream of Rho.
The Extracellular Domain of Kek1 Is Critical
for the Inhibitory Effect
To test whetherthe extracellulardomain of Kek1, which
contains fiveLRR andoneIg motif(Musacchioand Perri-
mon, 1996), is required for the inhibition of the EGFR
activity by Kek1, wegenerated transgenic lines thatcon-
tain either UAS-kek1extraor UAS-kek1intra.
No phenotype were observed by overexpressing
kek1intra(data not shown) in the follicle cells using the
T155 orCY2 GAL4 lines. On the otherhand, overexpres-
sion of kek1extrausing the same drivers led to eggs with
reduced dorsal appendage materials (Figure 5A) and
ventralized embryos (Figures 5B and 5C). These obser-
vations indicate that the extracellular domain of Kek1
is sufficient to inhibit the activity of the EGFR.
Although the phenotypes obtained following kek1extra
overexpression are very similar to those obtained with
full-length kek1, we note that these phenotypes are
weaker. One possibility is that Kek1extrais expressed at
a lower level than the WT protein. Weaker effects were
observed with 12 independent transgenic lines, as well
as when multiple copies of the transgenes were added.
This suggests that Kek1extrais less stable than the full-
length Kek1 molecule or that the cytoplasmic domain
of Kek1 participates to some extent in the inhibition.
Finally, we tested whether the inhibition of the EGFR
activity by kek1extrarequires the presence of the endoge-
nous Kek1 protein. When kek1extrawas overexpressed
excludes a model whereby kek1extrablocks the EGFR
by forming a heterodimer with the endogenous Kek1
protein (data not shown).
Figure 3. Kek1 Inhibits the Activity of the Ras/Raf/MEK/MAPK Cas-
Expression of kek-lacZ in WT (A) and following ectopic overexpres-
sion of kek1 in the follicular cell epithelium using either the T155 (B)
or CY2 Gal4 (C) drivers. Only the most dorsal anterior patch of
follicle cells at stage 10 are still expressing kek-lacZ in (B), while
the expression of the reporter gene is completely absent in (C). The
genotypes of the egg chambers shown in (B) and (C) are UAS-
kek1/kek-lacZ; T155 and UAS-kek1; kek-lacZ/CY2, respectively.
All panels are dorsal views of stage 10 egg chambers stained with
is overridden by the constitutive activation of the EGFR
or Draf (Figure 4). In addition, we tested the interaction
between the transmembrane protein Rhomboid (Rho)
and Kek1. Rho has been proposed to play a role in
the activation of the EGFR (Schweitzer et al., 1995a;
Golembo etal., 1996a), and overexpressionofRho leads
to dorsalized eggshells (Ruohola-Bakeret al., 1993). We
find that overexpression of rho does not override the
Figure 4. Genetic Epistasis between Kek1,
Rho, and Constitutively Activated Forms of
Either D-Raf or the EGFR (?Top)
(A) Chorion phenotypes. (1) Eggs derived
from UAS-Kek1; UAS-Rho/T155 females are
strongly ventralized. (2) Eggs derived from
UAS-Kek1; UAS-?top/T155 females are dor-
salized and are similar to those laid by UAS-
?top/T155 females. Dorsalization is clearly
visible by the extraappendage materials de-
posited around the entire egg circumference.
(B) Summary table of the various genotypes
A Novel Regulator of the Drosophila EGF Receptor
only one copy ofP[grk]lay WT eggs (Neuman-Silberberg
and Schupbach, 1994; this study, data not shown). We
found that eggs laid by females carrying one copy of
P[grk] inthe absence ofkek1 are significantly dorsalized
(Figure 6B1): the spacing between the dorsal append-
ages is increased and the eggs are rounderand smaller.
Further, Twi expression that is normally expressed in
embryos laid by females carrying one copy of P[grk]
(n ? 168; data not shown) is repressed ventrally in 15%
(n ? 181) of the embryos derived from kek1 mutant
females that carry a single copy of P[grk]. When raised
at 29?C, 22% (n ? 348) of the embryos derived from
eggs laid by females carrying one copy of P[grk] in the
absence ofkek1 showed a dorsalized phenotype readily
detectable by aberrant Twi expression. We note that
there is a variability in the reduction of the Twi domain,
ranging from embryos in which Twi-expressing cells are
missing in the middle regions of the embryo to the most
severe cases in which only the poles express the Twi
protein (Figures 6B2 and 6B3).
Second, we reasoned thatifloss ofKek1activity leads
to a hyperactivation of EGFR activity, then loss of Kek1
activity should rescue a decrease in EGFR activity. We
tested this hypothesis by generating flies that simulta-
neously lacked Kek1 activity and had reduced activity
of the EGFR. Consistent with our hypothesis, loss of
Kek1 activity resulted inpartialsuppressionofthe EGFR
phenotype (Figures 6C1±6C3). Eggs laid by females mu-
tant for EGFR alone have a ventralized phenotype
(Schupbach, 1987).They exhibiteithernooronlya single
dorsal appendage, and the eggs are significantly longer
than WT (100%, n ? 852; Figures 6C1 and 6C2). In
contrast, the majority of eggs (95.5%, n ? 1103) laid by
the kek1?egfr?double mutant females have two dorsal
appendages and their length is shorter, consistent with
a reversion to a more WT phenotype (Figure 6C3).
Figure 5. The Extracellular Domain of Kek1 Is Sufficient to Inhibit
the Activity of the EGFR
Eggs derived from CY2; UAS-kek1extrafemales that express kek1extra
in follicle cells using the Gal4 line CY2 exhibit a ventralized eggshell
phenotype (A). Consistently, the embryos that develop from these
eggs show a ventralized cuticle phenotype (B) and a lateral expan-
sion of Twi expression (C). (A) is a dorsal view, and (B) and (C) are
Kek1 Is a Negative Regulator of the Grk/EGFR
Because of the severe inhibition of EGFR activity by
Kek1 revealed by the overexpression experiment, we
decided to reexamine the effect of loss of kek1 function
during oogenesis. We reasoned that a subtle egg mor-
phology phenotype may have been missed by simply
using fertility as an assay (Musacchio and Perrimon,
1996). Indeed, we found that the spacing between the
dorsal appendages of eggs derived from kek1 mutant
females was increased when compared to WT. Further,
these eggs were also mildly shorterand rounder(Figure
6A1), a phenotype consistent with a hyperactivation of
the Grk/EGFR pathway (Neuman-Silberberg and Schup-
bach, 1994). These features do not interfere with hatch-
ing rates and patterning that is consistent with the nor-
mal Twi expression found in kek1 mutant embryos (n ?
142). Interestingly, when kek1 mutant flies were raised
at 29?C, 5% (n ? 257) of the embryos derived from
kek1 mutant females showed a mild reduction in Twi
expression (Figures 6A2 and 6A3). We conclude that
loss of kek1 activity during oogenesis leads to mildly
To substantiate the functional relationship between
Kek1 and the Grk/EGFR signaling pathway during oo-
genesis, we examined the effects of a loss of Kek1
activity in two different genetic backgrounds. First we
tested if an increase in the level of Grk molecules can
generate a stronger phenotype in the absence of kek1.
Previously, Neuman-Silberberg and Schupbach (1994)
showed thatfemales carrying fourcopies ofa transgene
expressing grk (P[grk]), in addition to the two endoge-
nous copies, lay a significant fraction of partially or se-
verely dorsalized eggs. In contrast, females that carry
Kek1 Physically Associates with the EGFR
Having demonstrated that the in vivo role of kek1 is
consistent with the results obtained by overexpression,
we wanted to gain insight into the mechanism of inhibi-
tion of the EGFR by Kek1. To do so, we tested whether
EGFR and Kek1 physically associate. Following coex-
pressionofa Myc-tagged versionofKek1 and the EGFR
or the Drosophila Torso RTK in Sf9 cells, Kek1 was
immunoprecipitated from the cell lysates using an anti-
Myc antibody. Coprecipitation of the EGFR was ob-
served by probing the resulting blot with the anti-EGFR
antibody (Figure 7A), suggesting that Kek1 associates
physically with the EGFR and that this interaction is
responsible for the inhibitory effect. When both Kek1
and Torso were coexpressed in Sf9 cells, no Torso was
coprecipitated by Kek1 (Figure 7A), suggesting a selec-
tivity of Kek1 for binding to the EGFR.
Because the extracellular and transmembrane por-
tions of Kek1 are essential for the inhibitory effect, we
examined whether these portions of Kek1 are able to
bind to the EGFR. As shown in Figure 7B, the extracellu-
lardomain, but not the intracellulardomain, of Kek1 can
bind to the EGFR. Thus, we propose that the inhibitory
effect of the EGFR by Kek1 is mediated through direct
association of the extracellularand transmembrane do-
mains of Kek1 with the EGFR.
Figure 6. Phenotypes Associated with Loss of kek1 Function during Oogenesis
(A1±A3) At 25?C, eggs derived from kek1 mutant females showed a weak dorsalization phenotype that does not interfere with fertility. This
dorsalization phenotype is evident by the more lateral position of the dorsal appendages (cf. Figure 6A1 with a WT eggshell shown in Figure
2A1). By increasing the temperature, we observed a weak enhancement of both the eggshell and the embryonic phenotypes readily detectable
by aberrant Twi expression (Figures 6A2 and 6A3).
(B1±B3) The dorsalization phenotype associated with loss of kek1 function is enhanced by an increase in the level of Grk ligand. The eggs
derived from kek1 mutant females that contain an extra copy of grk, provided by a P[grk] transgene, showed an enhancement of the dorsalized
eggshell phenotype (cf. Figure 6B1 with Figure 6A1), and the embryos derived from these eggs showed a stronger dorsalized phenotype (cf.
Figures 6B2 and 6B3 with Figures 6A2 and 6A3). Orientations are the same as in Figure 2.
(C1±C3) Suppression of the egg phenotype associated with egfr mutations by kek1. Chorions derived from egfr?or kek1?egfr?females are
shown. (C1) egfrQY1/egfr2E07. (C2) egfrQY1/kek1RM2egfr2E07. (C3) kek1RA5egfrQY1/kek1RM2egfr2E07. In addition to the recovery of the two dorsal
appendages, the length of the chorion has also become similar in length to WT.
Discussion The Function of Kek1 in Patterning of the Follicle
During oogenesis, Grk derived fromthe oocyte activates
in a paracrine fashion the EGFR in dorsal follicle cells.
Recent work from Wasserman and Freeman (1998) has
shown that this paracrine signaling leads to the activa-
tion of a second phase of signaling, whereby the EGFR
activity is amplified among follicle cells themselves.Dur-
ing this second phase, the EGFR activates a number of
target genes that include both positive (rho and vn) and
negative (aos) regulators of the pathway. Activation of
Rho in follicle cells presumably leads to the activation
of the Spi EGFR ligand, while activation of Aos within
the peak of EGFR activity at the dorsal anterior leads
to repression of EGFR, effectively splitting the initial
peak of EGFR activity into two. This splitting of EGFR
activity eventually defines the domains where the dorsal
appendages will form.
Our identification of Kek1, together with the studies
on Aos, indicates that there are at least two different
negative regulators of EGFR activity in follicle cells.
However, the regulation and function of Kek1 is distinct
from those of Aos. aos is expressed only in response
to high levels of EGFR activity, while kek1 is expressed
in a graded fashion. Further, loss-of-function pheno-
types of kek1 and aos in follicle cells are different. In
the absence of kek1 activity, the spacing between the
dorsal appendages is increased, while in the absence
We have identified the transmembrane protein Kek1 as
a molecule that acts in a negative feedback loop to
modulate the activity of the Drosophila EGFR tyrosine
kinase during oogenesis. We provide loss-of-function,
ectopic overexpression, and biochemical evidence to
support this. First, eggs laid by females that lack kek1
gene activity are weakly dorsalized, and loss of kek1
gene activity can suppress egfr mutations and potenti-
ate Grk signaling. Second, ectopic overexpression of
Kek1 infollicle cells leads to severe ventralizationidenti-
cal to effects seen in EGFR mutants, providing further
support of Kek1 as a negative regulator of the EGFR
pathway. Finally, we provide biochemical evidence that
theinhibitionofEGFR activitybyKek1involves thedirect
association between the extracellular and transmem-
brane domains of Kek1 and the EGFR.
Interestingly, kek1 is expressed in the eye and wing
imaginal discs in a pattern that is highly suggestive of
inductionby the EGFR (Musacchio and Perrimon, 1996).
Although no obvious phenotypes have been described
in these tissues in kek1 mutants, we found that overex-
pression of kek1 in these tissues also generate pheno-
types reminiscent to loss of EGFR activity (not shown).
This suggests that the negative regulation of the EGFR
by Kek1 is not only restricted to oogenesis.
A Novel Regulator of the Drosophila EGF Receptor
Further, we have shown that the extracellularand trans-
membrane domains of Kek1 are sufficient forthis inhibi-
tion. The extracellular domain of Kek1 contains one Ig-
like domain and five LRRs, both of which can mediate
protein±protein interactions (Musacchio and Perrimon,
1996).A numberofmechanisms canunderliethemecha-
nismby whichthis extracellulardomainacts as aninhibi-
tor. For example, the Kek1 extracellular domain could
mask the accessibility of the extracellular domain of
the EGFR to all ligands. Conversely, it could form
a heterodimer with EGFR monomers and block their
dimerization. Dimerization is a prerequisite to the ac-
tivation of downstream signaling events by the RTK.
Alternatively, Kek1 could be involved inbringing a trans-
membrane tyrosine phosphatase to the vicinity of the
EGFR and thus lead to its deactivation. Consistent with
these models, we have observed that Kek1 can inhibit
mammalian EGFR molecules from becoming tyrosine
phosphorylated in response to growth factor treatment
ininfected insect cells (L. T. A. et al., unpublished obser-
vations). Finally, we envision that Kek1 could target the
EGFR to a degradation pathway through endocytosis
orbind toadditionalproteins involved intheirsubcellular
localization. Studies on the C. elegans LET-23 EGFR
have well illustrated the critical role of subcellular local-
ization and PDZ proteins in signaling by this RTK (Kim,
1997; Kaech et al., 1998). Interestingly, we note that
both Kek1 and the EGFR contain a TXV motif at the
C terminus. This S/TXV motif has been implicated in
protein±protein interactions and suggests that Kek1
and/orthe EGFR may interact with PDZ-containing pro-
teins (Songyang et al., 1997; see reviews by Kim, 1997
and Ponting et al., 1997). Consistent withthis, ouranaly-
sis of the Kek1 molecule has led us to conclude that
Kek1extrais less stable than the full Kek1 molecule orthat
the cytoplasmic domain of Kek1 participates to some
extent in the inhibition. Possibly, Kek1 is targeted to a
subcellularregion within the cells where the EGFR itself
is located. This model would explain why unlocalized
Kek1extrawould be less efficient at inhibiting EGFR activ-
ity than the full-length Kek1 molecule.
Figure 7. Association of Kek1 with the Drosophila EGFR
(A) Lysates from Sf9 insect cells expressing the Drosophila EGFR
or Torso, or Kek1-Myc (kek) alone, or coexpressing Kek1-Myc with
each of the RTKs were immunoprecipitated with anti-Myc antibod-
ies. Precipitates were immunoblotted with anti-EGFR (upper left
panel) or anti-Torso (upper right panel) and reprobed with anti-Myc
(lower panels). In Sf9 cells, the EGFR migrates at two prominent
forms. The lowerEGFR band probably represents an underglycosy-
lated form of the receptor. We noted that at higher Kek1 expression
levels binding to Torso was also observed (not shown), perhaps
reflecting some degree of stickiness of the Kek1 protein.
(B)Sf9 cells were infected withbaculovirus encoding the Drosophila
EGFR and coinfected with nothing (None) or viruses encoding Myc-
tagged versions of full-length Kek1 (kek), a form encompassing the
transmembrane and the extracellular domains of Kek1 (kek ECD),
and a form encoding the intracellular domain of Kek1 fused to a
v-Src myristylation site (kek ICD). Anti-Myc immunoprecipitates
were blotted with anti-EGFR (left panel) and then reprobed with
anti-Myc (rightpanel).Notethatthe 55kDa Kek1intracellulardomain
is hidden in the Myc immunoprecipitate by the heavy chain of the
precipitating antibody, but it has been detected in the lysates.
An Emerging Theme in Patterning Mechanisms
Kek1 identifies a novel member of a growing class of
negative regulators that are positively transcriptionally
regulated by the pathway that they inhibit. As discussed
in the Introduction, the secreted molecule Aos acts in
a feedback loop to downregulate in a paracrine fashion
the activity of the EGFR, possibly by preventing the
(Schweitzer et al., 1995b; Golembo et al., 1996b). An-
other inhibitor of an RTK pathway is Sprouty, which
antagonizes the activity of the FGF receptor Breathless
(Btl) and is transcriptionally controlled by activated Btl
(Hacohen et al., 1998).
It is worthnoting, however, that not all secreted nega-
tive regulators of specific signaling pathways work in
negative feedback loops. These include Chordin/SOG
(Piccolo et al., 1997) and Noggin (Zimmerman et al.,
1996), whichantagonizesignaling by TGF-?family mem-
bers; FrzB proteins, which encode secreted proteins
with sequence homologies to the extracellularcysteine-
rich domain of Frizzled transmembrane receptors, act
ofaos, the appendages are fused dorsally.Aos has been
proposed to split the initial peak of EGFR activity into
two (Wasserman and Freeman, 1998). We propose that
the function of Kek1 is to restrict the lateral spreading
of EGFR activation by Spi. Thus, in the absence of kek1
activity, Rho/Spi activation could spread more laterally,
explaining the enhancement of the spacing betweenthe
two dorsal appendages.
Mechanism of Inhibition of the EGFR by Kek1
Our analysis demonstrates that Kek1 acts as a potent
negative regulator of EGFR activity when overexpressed.
The hypomorphic Draf mutation DrafHM7is described in Melnick et
al. (1993). hs-Drafgof-F22that expresses an N-terminal truncation of
Draf is described in Brand and Perrimon (1994). The UAS-EGFRDN
is described in Freeman (1996). The enhancer trap line es(3)79, the
P[grk], and the UAS-?top (Queenan et al., 1997) flies were a gift
from T. Schupbach.
Ectopic expressioninthe follicularepitheliumwas performed with
the pGawB GAL4lines T155(Harrisonetal., 1995)and CY2(Queenan
et al., 1997).
as inhibitors of secreted Wnt ligands (Leyns et al., 1997;
Wang etal., 1997).Finally,notallnegativeregulators that
act in feedback loops act extracellularly. For example,
Smad7, whichis expressed inresponse to TGF-?signal-
ing, blocks the activity of this pathway intracellularly by
binding to the TGF-? receptor and by inhibiting Smad2
and Smad3 phosphorylation that is required for trans-
duction of the signal (Nakao et al., 1997). Anotherexam-
ple is the human CL100 phosphatase that after being
expressed following exposure to either oxidative stress
or heat shock downregulates specifically the MAP ki-
nase (Keyse and Emslie, 1992). The Drosophila homolog
ofCL100, known as puckered, has beenshown to medi-
atea feedback loop regulating J NK activity during dorsal
closure in Drosophila (Martin-Blanco et al., 1998).
The colchicine inhibitor was used at 25 or 50 ?g/ml to destabilize
microtubules as described in Theurkauf et al. (1993). As a control,
flies were treated with 50 ?g/ml of ?-lumicolchicine, which is an
inactive isomer of colchicine. After initiating treatment, flies were
dissected and fixed at 4, 8, 10, and 24 hr intervals over 48 hr and
probed for expression of grk mRNAs. Stage 10 chambers treated
after the dorsal±anterior migration of the oocyte nucleus showed a
higherfrequency ofdisrupted and irregularkek-lacZ expression(49/
156) than chambers treated prior to the migration (17/108). Earlier
treated chambers had a higher frequency of posteriorly shifted gra-
dients (88/108 early versus 31/156 late). Finally, we detected no
effects with cytochalasin D, an inhibitor of microfilaments.
Kek1 Defines a Novel Gene Family
In Drosophila, two other putative Kek molecules that
share extensive homologies with kek1 have been identi-
fied: kek2 (Musacchio and Perrimon, 1996) and kek3
(our unpublished data). The function of these additional
Kek-like proteins is not known. However, it is interesting
to note that despite extensive saturation of the region
containing kek3, no mutant alleles have been recovered
(Ashburneretal., 1990;Spradling etal., 1995;ourunpub-
lished data). Whether or not this and the subtle effect
of loss of Kek1 activity is due to redundancy within the
Kekkon family remains to be determined. Thus, it will
be important to characterize the expression patterns as
wellas the loss-of-functionand overexpresssionpheno-
types ofkek2 and kek3 inorderto evaluate any potential
abilities to modulate the EGFR activity.Further, chimeric
proteins between these molecules may help to further
define the Kek1 domain(s) required for the inhibitory
Finally, we note that there are putative transmem-
brane proteins in vertebrates (Suzuki et al., 1996) and
invertebrates that show similar arrangements of LRRs
and Ig motifs. This raises the possibility that Kek1 is a
member of a family of structurally related EGFR inhibi-
tors. As alteration in the activity of the various members
of the human EGFR/ErbB family has strong links to on-
cogenesis, it will be important to determine if vertebrate
LRR/Ig molecules share functional, in addition to se-
quence, similarities to Kek1. We anticipate that the con-
tinued characterizationofthe Kek and related molecules
will provide novel approaches to the design of inhibitors
of the EGFR/ErbB family for therapeutic use in onco-
In Situ Hybridization and ?-Galactosidase Histochemistry
In situ hybridization was performed with digoxigenin-labeled grk,
kek1, and twist RNA essentially as described in Tautz and Pfeifle
(1989) using 55?C and 65?C as the hybridization temperature.
For detection of ?-galactosidase activity, ovaries were dissected
in PBT (PBS ? 0.1% Tween 20) and fixed in 4% methanol-free
formaldehyde for 15 min or in 2.5% glutaradehyde for 2.5 min. Ova-
ries were then stained with 1 mg/ml X-Gal in X-Gal staining buffer
at 25?C for 3.5±4.5 hr.
The polyclonal rabbit anti-Twist antibody (Roth et al., 1989) was
used at a dilution of 1:5000.
For visualization, embryos and egg chambers were directly
mounted in 80% glycerol and photographed underNomarski optics
with a Zeiss Axiophot microscope. Embryonic cuticles and chorions
were prepared according to van der Meer (1977) and visualized
using dark-field optics.
UAS-kek1 was made in the following manner. A HindIII/EcoRI frag-
ment from the kek1 cDNA containing the complete coding region
(Musacchio and Perrimon, 1996) was subcloned into pBSK?. From
this subclone, an XbaI/XhoIfragment containing the complete kek1
coding region was cloned into the P element vector pUAST (Brand
and Perrimon, 1993). UAS-kek1extrawas made by inserting by PCR
a stop codon immediately after the transmembrane domain of kek1
and UAS kek1intraby fusing a v-Src myristylation site to the entire
intracellular domain of kek1. P element±mediated transformation
was performed following injection into the delta 2±3 transposase
strain (Robertson et al., 1988).
Insect Cell Experiments
cDNAs encoding the Drosophila EGFR and myc-tagged versions
at the C terminus of full-length kek1, kek1extra, and kek1intrawere
subcloned into the baculovirus transfervectors pVL1392 orpVL1393
Recombinant baculoviruses were produced in Sf9 insect cells
using the Bac-N-Blue transfection kit (Invitrogen) and plaque puri-
fied prior to use. For coexpression experiments, 107Sf9 cells were
infected at a multiplicity of infection of 30. For the EGFR/Torso
comparison experiment, cells were infected with a 1:1:28 ratio of
Drosophila EGFR:Kek1:WT baculovirus. For the domain deletion
experiment, cells were infected with a 10:20 ratio of EGFR:Kek1
virus. For both experiments, cells were infected for 48 hr and then
lysed in 1 ml 20 mM HEPES/Na, 150 mM Nacl, 1mM EDTA, 1% NP-
40, 1 mM PMSF, and 1 mg/ml each of pepstatin A, aprotinin, and
leupeptin. Cleared lysates were immunoprecipitated with 1 ?g anti-
Myc (Ab2, NeoMarkers). Precipitates were washed three times with
lysis buffer, resolved by 6% SDS-PAGE, and blotted with 1/5000
Various collections ofenhancertrap lines were screened forexpres-
sion patterns within the follicular epithelium (Perrimon et al., 1991;
Smith et al., 1993; D. Eberl, unpublished). From this screen, the
second chromosomal enhancer trap line 15A6 was identified. For a
description of 15A6, which we refer to as kek-lacZ, see Musacchio
and Perrimon (1996). The two overlapping deficiencies, RA5 and
RM2, completely delete the kek1 gene (Musacchio and Perrimon,
1996), and we refer to RA5/RM2 females as kek1 mutant females
in this paper.
The alleles of the egfr, egfrQYI, egfrCJ, egfr101, egfr2E07are described
in Clifford and Schupbach (1989). For a description of grkHK36, see
Schupbach (1987) and Neuman-Silberberg and Schupbach (1993).
A Novel Regulator of the Drosophila EGF Receptor
anti-EGFR or 1/5000 anti-Torso. Filters were then stripped and re-
probed with 1/200 anti-Myc.
Kharitonenkov, A., Chen, Z., Sures, I., Wang, H., Schilling, J ., and
Ullrich, A. (1997). A family of proteins that inhibit signaling through
tyrosine kinase receptors. Nature 386, 181±186.
Kim, S.K. (1997). Polarized signaling: basolateral receptor localiza-
tion in epithelial cells by PDZ-containing proteins. Curr. Opin. Cell
Biol. 9, 853±859.
Kokel, M., Borland, C.Z., DeLong, L., Horvitz, H.R., and Stern, M.J .
(1998). clr-1 encodes a receptor tyrosine phosphatase that nega-
tively regulates anFGF receptorsignaling pathway incaenorhabditis
elegans. Genes Dev. 12, 1425±1437.
Leyns, L., Bouwmester, T., Kim, S.H., Piccolo, S., and De Robertis,
E.M. (1997). Frzb-1 is a secreted antagonist of Wnt signaling ex-
pressed in the Spemann organizer. Cell 88, 747±756.
Li, W., and Perrimon, N. (1997). Specificity of receptor tyrosine sig-
naling pathways: lessons fromDrosophila. InGenetic Engineering V.
19: Principles and Methods (New York: Plenum Press), pp. 167±182.
Martin-Blanco, E., Gampel, A., Ring, J ., Virdee, K., Kirov, N., Tolkov-
sky, A.M., and Martinez-Arias, A. (1998). puckered encodes a phos-
phatase that mediates a feedback loop regulating J NK activity dur-
ing dorsal closure in Drosophila. Genes Dev. 12, 557±570.
Melnick, M.B., Perkins, L.A., Lee, M., Ambrosio, L., and Perrimon,
N. (1993). Developmental and molecular characterization of muta-
tions in the Drosophila raf serine-threonine kinase. Development
Morisato, D., and Anderson, K.V. (1995). Signaling pathways that
establishthe dorsal-ventralpatternof the Drosophila embryo. Annu.
Rev. Genet. 29, 371±399.
Musacchio, M., and Perrimon, N. (1996). The Drosophila kekkon
genes: novel members of both the leucine-rich repeat and immuno-
globulin superfamilies expressed in the CNS. Dev. Biol. 178, 63±76.
Nakao, A., Afrakhte, M., Moren, A., Nakayama, T., Christian, J .L.,
Heuchel, R., Itoh, S., Kawabata, M., Heldin, N.E., Heldin, C.H., and
tenDijke, P.(1997).IdentificationofSmad7, a TGF?-inducible antag-
onist of TGF-? signaling. Nature 389, 631±635.
Neuman-Silberberg, F.S., and Schupbach, T. (1993). The Drosophila
dorsoventral patterning gene gurken produces a dorsally localized
RNA and encodes a TGF?-like protein. Cell 75, 165±174.
Neuman-Silberberg, F.S., and Schupbach, T. (1994). Dorsoventral
axis formation in Drosophila depends on the correct dosage of the
gene gurken. Development 120, 2457±2463.
Perrimon, N., and Perkins, L.A. (1997). There must be 50 ways to
rule the signal: the case of the Drosophila EGF receptor. Cell 89,
Perrimon, N., Noll, E., McCall, K., and Brand, A. (1991). Generating
lineage-specific markers to study Drosophila development. Dev.
Genet. 12, 238±252.
Piccolo, S., Agius, E., Lu, B., Goodman, S., Dale, L., and De Robertis,
E.M. (1997). Cleavage of Chordin by Xolloid metalloprotease sug-
gests a role for proteolytic processing in the regulation of Spemann
organizer activity. Cell 91, 407±416.
Ponting, C.P., Philips, C., Davies, K.E., and Blake, D.J . (1997). PDZ
domains: targeting signaling molecules to sub-membranous sites.
Bioessays 19, 469±479.
Price, J .V., Clifford, R.J ., and Schupbach, T. (1989). The maternal
ventralizing locus torpedo is allelic to faint little ball, an embryonic
lethal, and encodes the Drosophila EGF receptor homolog. Cell 56,
Queenan, A.M., Ghabrial, A., and Schupbach, T. (1997). Ectopic
activation of torpedo/Egfr, a Drosophila receptor tyrosine kinase,
dorsalizes both the eggshell and the embryo. Development 124,
Ray, R.P., and Schupbach, T. (1996). Intercellular signaling and the
polarization of body axes during Drosophila oogenesis. Genes Dev.
Robertson, H.M., Preston, C.R., Phillis, R.W., J ohnson-Schlitz, D.,
Benz, W.K., and Engels, W.R. (1988). A stable genomic source of
P element transposase in Drosphila melanogaster. Genetics 118,
Roth, S., and Schupbach, T. (1994). The relationship between ovar-
ian and embryonic dorsoventral patterning in Drosophila. Develop-
ment 120, 2245±2257.
We thank M. Musacchio and Z. Wills for their contributions to the
early phase of this project; C. Arnold for injecting constructs; A. J .
Diamonti for his expert technical assistance; and A. Brand, V.
Cleghon, B. Dickson, D. Eberl, A. Michelson, S. Roth, H. Ruohola-
Baker, T. Schupbach, and B. Shilo forreagents. C. G was supported
by an EMBO fellowship, and J . B. D. was supported by a Damon
Runyon±WalterWinchellCancerFoundationFellowship (DRG 1213).
L.T. A.was supported by a Howard Hughes postdoctoralfellowship.
R. E. B. is an Assistant Investigator of the Howard Hughes Medical
Institute. N. P. is an Investigator of the Howard Hughes Medical
Institute.This work was supported inpartby US Army grants (K.L.C.
and N. P.) and by NIH grant CA71702 (to K. L. C.).
Received November 20, 1998; revised February 16, 1999.
Ashburner, M., Thompson, P., Roote, J ., Lasko, P.F., Grau, Y., El
Messal, M., Roth, S., and Simpson, P. (1990). The genetics of a
small autosomal region of Drosophila melanogaster containing the
structural gene for alcohol dehydrogenase. VII. Characterization of
the region around the snail and cactus loci. Genetics 126, 679±694.
Brand, A., and Perrimon, N. (1993). Targeted gene expression as a
means of altering cell fates and generating dominant phenotypes.
Development 118, 401±415.
Brand, A.H., and Perrimon, N. (1994). Raf acts downstream of the
EGF receptor to determine dorsoventral polarity during Drosophila
oogenesis. Genes Dev. 8, 629±639.
Clifford, R.C., and Schupbach, T. (1989). Coordinately and differen-
tially mutable activities of torpedo, the Drosophila melanogaster
homologue of the vertebrate EGF receptor gene. Genetics 123,
Freeman, M. (1996). Reiterative use of the EGF receptor triggers
Freeman, M., Klambt, C., Goodman, C.S., and Rubin, G.M. (1992).
The argos gene encodes a diffusible factor that regulates cell fate
decisions in the Drosophila eye. Cell 69, 963±975.
Golembo, M., Raz, E., andShilo, B.Z.(1996a).The Drosophila embry-
onic midline is the site of Spitz processing, and induces activation
of the EGF receptor in the ventral ectoderm. Development 122,
Golembo, M., Schweitzer, R., Freeman, M., and Shilo, B.Z. (1996b).
Argos transcriptionis induced by the Drosophila EGF receptorpath-
way to formaninhibitory feedback loop. Development122, 223±230.
Greenwood, S., and Struhl, G. (1997). Different levels of Ras activity
can specify distinct transcriptional and morphological conse-
quences inearly Drosophila embryos.Development124, 4879±4886.
Hacohen, N., Kramer, S., Sutherland, D., Hiromoi, Y., and Krasnow,
M.A. (1998). Sprouty encodes a novel antagonist of FGF signaling
that patterns apical branching of the Drosophila airways. Cell 92,
Harrison, D.A., Binari, R., Nahreini, T.S., Gilman, M., and Perrimon,
N. (1995). Activation of a Drosophila janus kinase (J AK) causes he-
matopoietic neoplasia and developmental defects. EMBO J . 14,
Hsu, J .C., and Perrimon, N. (1994). A temperature sensitive MEK
mutation demonstrates the conservation of the signaling pathways
activated by receptor tyrosine kinases. Genes Dev. 8, 2176±2187.
Kaech, S.M., Whitfield, C.W., and Kim, S.K. (1998). The LIN-2/LIN-7/
LIN-10 complex mediates basolateral membrane localization of the
C. elegans EGF receptor LET-23 in vulval epithelial cells. Cell 94,
Keyse, S.M., and Emslie, E.A. (1992). Oxidative stress and heat-
shock induce a human gene encoding a protein-tyrosine phospha-
tase. Nature 359, 644±647.
Cell Download full-text
Roth, S., Stein, D., and Nusslein-Volhard, C. (1989). A gradient of
nuclear localization of the dorsal protein determines dorsoventral
pattern in the Drosophila embryo. Cell 59, 1189±1202.
Ruohola-Baker, H., Grell, E., Chou, T.B., Baker, D., J an, L.Y. and
J an, Y.N. (1993). Spatially localized rhomboid is required for estab-
lishment of the dorsal-ventral axis in Drosophila oogenesis. Cell 73,
Rutledge, B., Zhang, K., Bier, E., J an, Y.N., and Perrimon, N. (1992).
TheDrosophilaspitz geneencodes aputative EGF-likegrowthfactor
involved in dorsal-ventral axis formation and neurogenesis. Genes
Dev. 6, 1503±1517.
Sapir, A., Schweitzer, R., andShilo, B.Z.(1998).Sequentialactivation
of the EGF receptor pathway during Drosophila oogenesis estab-
lishes the dorsoventral axis. Development 125, 191±200.
Schnepp, B., Grumbling, G., Donaldson, T., and Simcox, A. (1996).
Veinis a novelcomponent inthe Drosophila epidermalgrowthfactor
receptor pathway with similarity to the neuregulins. Genes Dev. 10,
Schnorr, J .D., and Berg, C.A. (1996). Differential activity of Ras1
during patterning of the Drosophila dorsoventral axis. Genetics 144,
Schupbach, T. (1987). Germline and soma cooperate during oogen-
esis to establishthe dorsoventralpatternofthe eggshelland embryo
in Drosophila melanogaster. Cell 49, 699±707.
Schweitzer, R., and Shilo, B.Z. (1997). A thousand and one roles for
the Drosophila EGF receptor. Trends Genet. 13, 191±196.
Schweitzer, R., Shaharabany, M., Seger, R., and Shilo, B.Z. (1995a).
Secreted Spitz triggers the DER signaling pathway and is a limiting
component in embryonic ventral ectoderm determination. Genes
Dev. 9, 1518±1529.
Schweitzer, R., Howes, R., Smith, R., Shilo, B.Z., and Freeman, M.
(1995b). Inhibition of Drosophila EGF receptor activation by the se-
creted protein Argos. Nature 376, 699±702.
Smith, D., Wohlgemuth, J ., Calvi, B.R., Franklin, I., and Gelbart, W.M.
(1993). Hobo enhancer trapping mutagenesis in Drosophila reveals
an insertion specificity different from P elements. Genetics 135,
Songyang, Z., Fanning, A.S., Fu, C., Xu, J ., Marfatia, S.M., Chishti,
A.H., Crompton, A., Chan, A.C., Anderson, J .M., and Cantley, L.C.
(1997). Recognition of unique carboxyl-terminal motifs by distinct
PDZ domains. Science 275, 73±77.
Speicher, S.,Garcia-Alonso, L., Carmena, A.,Martin-Bermudo, M.D.,
de la Escalera, S., and J imenez, F. (1998). Neurotactin functions
in concert with other identified CAMs in growth cone guidance in
Drosophila. Neuron 20, 221±233.
Spradling, A.C., Stern, D., Kiss, I., Roote, J ., Laverty, T., and Rubin,
G.M. (1995). Gene disruptions using P transposable elements: an
integral component of the Drosophila genome project. Proc. Natl.
Acad. Sci. USA 92, 10824±10830.
Sturtevant, M.A., O'Neill, J .W., and Bier, E. (1994). Down-regulation
of Drosophila EGF receptor mRNA levels following hyperactivated
receptor signaling. Development 120, 2593±2600.
Suzuki, Y., Sato, N., Tohyama, M., Wanaka, A., and Takagi, T. (1996).
cDNA cloning of a novel membrane glycoprotein that is expressed
specifically in glial cells in the mouse brain. LIG-1, a protein with
leucine-rich repeats and immunoglobulin-like domains. J . Biol.
Chem. 271, 22522±22527.
Tautz, D., and Pfeifle, C. (1989). A non-radioactive in situ hybridiza-
tion method for the localization of specific RNAs in Drosophila em-
bryos reveals translational control of the segmentation gene hunch-
back. Chromosoma 98, 81±85.
Theurkauf, W.E., Alberts, B.M., J an, Y.N., and J ongens, T.A. (1993).
A central role for microtubules in the differentiation of Drosophila
oocytes. Development 118, 1169±1180.
Thisse, B., Stoetzel, C., Gorostiza-Thisse, C., and Perrin-Schmitt, F.
(1988). Sequence of the twist gene and nuclear localization of its
protein in endomesodermal cells of early Drosophila embryos.
EMBO J . 7, 2175±2183.
Ullrich, A., and Schlessinger, J .(1990). Signaltransductionby recep-
tors with tyrosine kinase activity. Cell 61, 203±212.
van der Geer, P., Hunter, T., and Lindberg, R.A. (1994). Receptor
tyrosine kinases and theirsignal transduction pathways. Annu. Rev.
Cell Biol. 10, 208±251.
van der Meer, J . (1977). Optical clean and permanent whole mount
preparation for phase-contrast microscopy of cuticular structures
of insect larvae. Dros. Inf. Serv. 52, 160±170.
Wang, S., Krinks, M., Lin, K., Luyten, F.P., and Moos, M., J r. (1997).
Frzb, a secreted protein expressed in the Spemann organizer, binds
and inhibits Wnt-8. Cell 88, 757±766.
Wasserman, J .D., and Freeman, M. (1998). An autoregulatory cas-
cade of EGF receptor signaling patterns the Drosophila egg. Cell
Zimmerman, L.B., De, J ., Escobar, J .M., and Harland, R.M. (1996).
The Spemann organizer signal noggin binds and inactivates bone
morphogenetic protein 4. Cell 86, 599±606.