Department of Molecular Biology and Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8103, St Louis, MO 63110, USA
Accepted 15 May 2003
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SUMMARY |
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Key words: Drosophila, Echinoid, Egfr, R8, Retina
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INTRODUCTION |
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Similar to most sensory systems, the developing fly eye employs a variety of patterning mechanisms to produce a precise arrangement of differentiated cells. It is a composite of some 750 unit eyes, or `ommatidia', arranged into a perfect lattice, giving rise to the crystalline appearance of the adult compound eye. The precision with which this lattice forms makes the developing Drosophila retina an excellent system for identifying even modest alterations in important signaling pathways. Within the larval eye disc, the first cell to emerge within each ommatidium is the R8 photoreceptor. Differentiating R8s arise in rows within the morphogenetic furrow, each cell separated from its neighbor by 10-12 undifferentiated cells, and each row of cells precisely out of register with those in the row posterior to it (Fig. 1A-C). Once it emerges, each R8 cell begins recruitment of the other cell types necessary to form a mature ommatidium.
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The cells that comprise the proneural clusters exhibit high levels of
phosphorylated Rolled/ERKA/MAP kinase, which constitutes a readout of Egfr
activity. Rhomboid and Roughoid are activators of Egfr signaling that are
expressed by cells at the posterior edge of the proneural clusters, and are
responsible for the phosphorylated MAP kinase observed within these cells
(Wasserman et al., 2000). This
activation of the Egfr pathway appears to be short lived, coinciding precisely
with the expression of Atonal. This period of activation is essential for R8
patterning: both hyperactivation and loss of Egfr function disrupt R8
differentiation and/or spacing (Baker and
Rubin, 1989
; Freeman,
1996
; Dominguez et al.,
1998
; Spencer et al.,
1998
). Patches of developing eye tissue lacking Egfr activity show
a loss of well-defined proneural clusters, a loss of proper R8 spacing and an
overall decrease in the number of R8 cells as assessed by Boss expression
(Dominguez et al., 1998
).
Conversely, transient overactivation of Ras pathway signaling increases Atonal
expression throughout the morphogenetic furrow and prompts differentiation of
additional R8 cells (Spencer et al.,
1998
). This latter result highlights the importance of limiting
Egfr activity in the morphogenetic furrow to restrict the number of R8s.
The precise level and duration of Egfr activity is subject to several
levels of control. A number of inhibitors of Egfr have been described in
Drosophila, such as Argos
(Schweitzer et al., 1995),
Kekkon (Ghiglione et al.,
1999
) and Sprouty (Casci et
al., 1999
). Each of these factors is transcribed in response to
Egfr signaling, but none has been shown to play a role in limiting Egfr
activity during R8 development. Egfr signaling is also attenuated by
activity-dependent endocytosis, and proteins that control this process, such
as cbl, are essential for maintaining Egfr signaling of appropriate
duration (Levkowitz et al.,
1998
; Pai et al.,
2000
). In this paper, we present evidence that the transmembrane
protein Echinoid (Bai et al.,
2001
) plays an essential role in R8 selection and retinal
development by downregulating Egfr signaling after an initial period of
activation. We further demonstrate that Echinoid and Egfr associate with one
another and that Echinoid is a substrate for tyrosine phosphorylation in
response to Egfr activity, suggesting a direct role for Echinoid in limiting
Egfr signaling within the developing retina.
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MATERIALS AND METHODS |
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Constructs
echinoid (EST clone GM09285) was tagged with a FLAG epitope
(edFLAG) or Myc epitope (edmyc) at the C terminus and cloned
into the UAST vector for creation of transgenic flies, or into the heat-shock
CaSpeR vector for expression in cultured S2 cells. EdFLAGC, which lacks
the tyrosine-rich region of the Echinoid intracellular domain, was created by
deleting amino acids 1078-1332 and fusing a FLAG epitope to the C terminus.
EdFLAG
N, which lacks the Ig and FN3 domains (but not the signal
sequence), was created by deleting amino acids 69-787 and tagging the C
terminus with a FLAG epitope. Egfr in hs-CaSpeR (hs-Egfr) and secreted Spitz
in hs-CaSpeR (hssspi) were gifts from B. Shilo. Morgue-FLAG in pRMHa3 was a
gift from Rebecca Hays. ERK-myc in pPAC was a gift from Tina Tootle.
RasVal12 in pIE1-3 was a gift from Callie Craig.
Antibodies and immunohistochemistry
Antibodies directed against Atonal (used 1:5000, gift from Y. Jan), Boss
(used 1:2000, gift of Larry Zipursky), Senseless (used 1:1000, gift of H.
Bellen) and dually phosphorylated ERKA (used 1:500, ERK; Sigma) were used to
stain eye imaginal discs as described previously
(Spencer et al., 1998): eye
imaginal discs from third instar larvae were dissected into PBS and
transferred immediately to 4% paraformaldehyde in PBS for 20 minutes. After
washing twice in PBS and twice in PBS+0.3% TritonX-100 (Sigma), discs were
incubated 4 hours at 4 degrees in primary antibody, PBS + 0.3% Triton X-100,
0.5 mg/ml BSA. After washing three times in PBS+0.3% TritonX-100, discs were
further incubated in fluorescently tagged secondary antibodies [Cy3- or
FITC-tagged anti-mouse and anti-rabbit, Jackson Immunochemicals; Alexa568-anti
guinea pig (Santa Cruz) in PBS + 0.3% Triton X-100, 0.5 mg/ml BSA]. Antibodies
against Corkscrew (CT-1 and CT-2; gift of M. Simon), phosphotyrosine
(PY99-HRP; Santa-Cruz Biotechnology), Flag (HRP-linked M2; Sigma), Egfr (a
gift from Nick Baker), C-terminal Echinoid (a gift from E. Rawlins and A.
Jarman), N-terminal Echinoid (a gift from J.-C. Hsu) and Myc (rabbit
polyclonal A-15; Santa-Cruz) were used for immunoprecipitations and western
blot analysis.
Tissue culture and immunoprecipitations
S2 and SL2 cells were transiently transfected using Cellfectin (Cell
Signaling). Transfection efficiency was monitored using pIE-1-Bgal. The amount
of total heat-shock promoter in each transfection condition was kept constant
by adding empty hs-CaSpeR vector to some reactions. Twenty-four hours after
transfection, cells were heat-shocked three times for 30 minutes with 30
minute rests to prompt expression of genes in the hs-CaSpeR vector. Cells were
washed once with room temperature PBS and lysed on ice with NP-40 lysis
buffer: 1% NP-40, 50 mM Tris, pH 7.4, 1 mg/ml BSA, 1 mM Na-VO4, 1x
Complete Protease Inhibitor cocktail. Cells were lysed at 4°C with mixing
for 20 minutes, then centrifuged at 13,000 g for 15 minutes to
remove particulate matter. Lysates were pre-incubated with ProteinA/Protein G
Sepharose (Gamma-Bind, Pharmacia) for 30 minutes to reduce non-specific
binding. Immunoprecipitations were carried out on lysates for 2-12 hours at
4°C using Sepharose linked to FLAG-M2 monoclonal antibodies (Sigma) or
myc-A15 monoclonal antibodies (Santa Cruz). Beads were washed four times with
lysis buffer and once with lysis buffer lacking BSA before eluting bound
proteins with SDS-loading buffer. Novex 4-12% polyacrylamide gels were used
for SDS-PAGE. Western analysis was essentially as described previously
(Spencer et al., 1998);
antibodies were used at the following concentrations: against Corkscrew (CT-1
and CT-2, 1:2000), phosphotyrosine (PY99-HRP; 1:500), Flag (HRP-linked M2;
1:500), Egfr (rabbit; 1:2000), N-terminal Echinoid (1:1000) and Myc (rabbit
polyclonal A-15; 1:1000).
Immunoprecipitations from eye imaginal discs
Immunoprecipitations from tissue were carried out as described above with
the following changes. Twenty third-instar larvae were obtained from each
genotype: w1118, hs-rhomboid (animals containing
a transgene for heat-shock-induced rhomboid expression) or
hs-argos (heat-shock-induced argos expression). Larvae were
heat-shocked for 35 minutes in a 37°C water bath to induce transgene
expression. Eyeantennal imaginal discs were immediately dissected from the
animals in PBS and immediately transferred to NP-40 lysis buffer (described
above). Discs were dissociated by drawing lysates up in a syringe with a
22-guage needle four times; lysates were otherwise treated as described for
cultured cells.
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RESULTS |
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To examine the effect that loss of echinoid function had on R8 specification, we examined Atonal expression and R8 formation both in echinoid homozygotes and in eye discs mosaic for echinoid. In both cases, we found that R8 specification was disrupted. Atonal normally narrows from an equivalence group of two or three cells to a single cell posterior to the morphogenetic furrow. In genotypically echinoid discs, this narrowing fails to occur and Atonal remains present in small groups of cells as they emerge from the morphogenetic furrow (Fig. 2A,B). This defect is particularly conspicuous in young eye discs from early third instar animals in which the morphogenetic furrow has only advanced a short distance, although the defect is also widespread later in eye development and in clonal patches of cells lacking echinoid (Fig. 2C,D). We observed no defects, however, in the initial stages of Atonal expression, or in its resolution to proneural groups. We conclude from this that echinoid is required only for the resolution of the R8-equivalence group to a single cell.
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In adult fly eyes, the R8 photoreceptor is distinguished by a small inner rhabdomere, or light-gathering organ, in the bottom third of the retina. Although R7 cells also contain small rhabdomeres, these are visible only in more apical retinal sections. Consistent with the results in larval discs, genotypically echinoid tissue often contained multiple inner rhabdomeres in basal sections that did not extend through to more apical levels (Fig. 2K,L). R8 is the first cell to differentiate in the eye; it is required for the proper recruitment of the other photoreceptors and accessory cells that compose the mature ommatidium. The differentiation of more than one R8 cell within a single ommatidium disrupts the proper recruitment of other cell types; the result is often an enlarged or fused ommatidium containing more than the normal complement of eight photoreceptor neurons (Fig. 2K,L).
Our results contrast with those of a previous report where no defects in R8
specification were observed in echinoid mutants
(Bai et al., 2001). Bai et al.
report instead the presence of extra R7 cells in an echinoid
background. We examined sections of adult eyes containing clonal patches of
edl(2)k01102 tissue: in all clones where ommatidia had
multiple R7 cells, multiple R8 cells were present as well (n=11
clones). R8 induces the R7 fate (Van
Vactor et al., 1991
); our evidence, therefore, cannot distinguish
whether these ectopic R7 cells are due solely to induction by ectopic R8 cells
or are also a direct consequence of the echinoid mutation.
Loss of echinoid leads to sustained ERKA activation
One of the first hallmarks of R8 formation within the MF is the appearance
of proneural clusters containing high levels of Atonal
(Jarman et al., 1994;
Sun et al., 1998
). These
groups of six to ten cells also contain high levels of ERKA phosphorylation
(Kumar et al., 1998
;
Spencer et al., 1998
)
(Fig. 3A,B), a readout of
Ras1-pathway signaling (Gabay et al.,
1997
). As the morphogenetic furrow progresses anteriorly, ERKA
phosphorylation is normally lost and the proneural clusters resolve to single
Atonal-expressing cells. By contrast, in echinoid discs a high level
of ERKA phosphorylation was retained in small groups of cells even after the
morphogenetic furrow had moved anteriorly
(Fig. 3C-F).
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echinoid and argos both restrict Egfr signaling
during R8 development
Argos is another inhibitor of Egfr present in the eye disc during R8
formation. However, analysis of tissue lacking argos shows few
defects in R8 patterning (Baonza et al.,
2001; Freeman et al.,
1992
; Spencer et al.,
1998
), suggesting that another factor may be acting redundantly.
To test whether Echinoid and Argos may be acting together to inhibit Egfr
signaling in the morphogenetic furrow, we examined their ability to interact
in vivo. Removing one copy of argos
(argos
7) mildly enhanced the
edl(2)k01102 phenotype: the number of ommatidia with
multiple R8 cells increased from 18% to 34%, as assessed with antibodies
against Boss (n=412). The viable hypomorphic argos mutation,
argosW11, also enhanced the echinoid rough eye
phenotype (Fig. 4A-D): the
number of ommatidia with multiple R8s increased from 21% to 46%
(n=336). This enhancement is presumably caused by a progressive
increase in Egfr activity. However, Echinoid and Argos differ in their mode of
action. Egfr inhibitors such as Argos and Kekkon are expressed in response to
high levels of Egfr signaling to form negative feedback loops
(Ghiglione et al., 1999
;
Schweitzer et al., 1995
).
Echinoid, by contrast, is ubiquitously expressed in the eye disc
(Bai et al., 2001
) (S.S. and
R.C., unpublished), suggesting that it is not transcriptionally regulated by
Egfr activity, which acts within discreet regions of the eye disc
(Fig. 3A). Furthermore, unlike
Argos, overexpression of Echinoid in the morphogenetic furrow does not lead to
defects in ERKA phosphorylation or R8 specification
(Fig. 4E-H). Our data indicate
that specific Egfr signaling is not sensitive to relatively large changes in
the amount of Echinoid present, and implies that if the activity of Echinoid
is regulated, it is by a means other than transcription.
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Echinoid associates with Egfr
Echinoid has been proposed to act in a pathway parallel to Egfr, with
signaling converging at the nucleus (Bai et
al., 2001). Our data suggests that loss of echinoid acts
upstream of the nucleus to stabilize ERKA phosphorylation. Given the
localization of Echinoid at the cell surface
(Bai et al., 2001
) (data not
shown) we explored the potential for direct interactions between Echinoid and
Egfr in S2 cultured cells. FLAG-tagged Echinoid was immunoprecipitated and
analyzed by western blots: transfection with increasing amounts of Echinoid
co-precipitated increasing amounts of Egfr, suggesting that this is a specific
interaction (Fig. 5C).
Similarly, Echinoid containing a Myc tag also co-precipitated Egfr; other
FLAG- and Myc-tagged proteins did not (Fig.
5C). In the converse experiment, immunoprecipitation of Egfr
co-precipitated EdFLAG (Fig.
5D). The site of binding between Echinoid and Egfr could not be
shown conclusively from these experiments: both EdFLAG
C and
EdFLAG
N efficiently immunoprecipitated Egfr (data not shown),
suggesting that their interaction occurs near the transmembrane domain or
through multiple domains.
Echinoid is phosphorylated in response to Egfr activity
L1 cell-adhesion proteins are frequently regulated by phosphorylation,
which controls their binding to other cytoplasmic signaling proteins. The
physical association of Echinoid with Egfr, a receptor tyrosine kinase,
suggested that it might be a substrate for tyrosine phosphorylation. To
examine this possibility, we used S2 cell culture to assess tyrosine
phosphorylation in response to Egfr signaling. As seen in
Fig. 6A, Echinoid exhibited a
low level of tyrosine phosphorylation in the absence of added Egfr.
Co-transfection with Egfr, even without added ligand, led to a dramatic
increase in the phosphorylation of Echinoid. This phosphorylation was
increased further by addition of media containing a soluble form of the
activating Egfr ligand Spitz. As seen in
Fig. 6B, phosphorylation was
limited to the tyrosine-rich intracellular region: a form of Echinoid lacking
the intracellular domain (EdFLAGC; see
Fig. 5E) was not
phosphorylated. Interestingly, EdFLAG
N, which lacks the extracellular
immunoglobulin and fibronectin motifs, was phosphorylated in response to Egfr
signaling, suggesting that these extracellular motifs are unnecessary for
phosphorylation to occur. Bai et al. (Bai
et al., 2001
) suggested that Echinoid acts in a pathway parallel
to Egfr, and that their activities converge in the nucleus. We find, however,
that expression of the activated Ras isoform dRas1Val12 did not
lead to increased Echinoid phosphorylation (data not shown), indicating that
phosphorylation of Echinoid in response to Egfr signaling occurs upstream of
Ras activation. These data do not resolve whether Echinoid is phosphorylated
directly by Egfr or by cytoplasmic tyrosine kinases immediately downstream of
its activity, an issue also unresolved for other cell-adhesion proteins.
Phosphorylation of Echinoid in response to Egfr signaling was confirmed in
vivo: Echinoid immunoprecipitated from w1118 eye discs
(which are essentially wild type at this stage) show a low level of
phosphorylation (Fig. 6C).
Echinoid immunoprecipitated from discs in which Rhomboid (an activator of
Egfr) was transiently expressed shows an approximately fourfold increase in
tyrosine phosphorylation. Transient expression of Argos, an inhibitor of Egfr,
produced either no change or a slight decrease in Echinoid phosphorylation
levels over wild type. The importance of this phosphorylation to the function
of Echinoid is not clear from these data. It has been noted, however, that
strong overexpression of Echinoid in the developing retina leads to a rough
eye in adults (Bai et al.,
2001
). This phenotype requires the presence of the Echinoid
intracellular domain (Bai et al.,
2001
), suggesting that this domain may be essential for the
function of Echinoid.
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DISCUSSION |
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R8 patterning reflects at least two processes: spacing of emerging R8
equivalence groups and selection from these groups of single R8 cells. We
previously suggested that expression of Egfr inhibitors is important for
setting the spacing between R8 cells
(Spencer et al., 1998), a view
supported by mispatterning in loss-of-function Egfr clones (Dominguez and
Freeman, 1998). We find, however, that echinoid plays no role in this
process: while loss of echinoid does increase the duration of Egfr
signaling, it does not affect the initial pattern of Egfr activity or the
position of R8 equivalence groups within the morphogenetic furrow. Rather,
Echinoid appears to be essential only for the second step in R8 specification,
the selection of a single R8 cell from the 2-3 cell equivalence group. The
role of Echinoid is to ensure that Egfr activity is downregulated within the
group in a timely fashion; persistent Egfr activation appears to trigger all
cells of the equivalence group to differentiate as R8s. Consistent with this,
expression of an activated-Ras (Spencer et
al., 1998
), activated-Raf or Pointed-P1
(Rawlins et al., 2003
) promote
multiple R8 cells within individual ommatidia.
Interestingly, Echinoid is the second example of a co-factor required for
fine-tuning a major signaling pathway during R8 selection. Selection of R8
from the equivalence group also requires scabrous, a modifier of
Notch signaling (Cagan, 1993;
Dokucu et al., 1996
;
Powell et al., 2001
). Egfr and
Notch signaling are used in a number of developing tissues. Echinoid and
Scabrous appear to fill the need for high precision during resolution of the
R8 equivalence group; this precision is almost unique in the developing
nervous system. Therefore, Echinoid and Scabrous appear to have evolved to
fine-tune these two pathways for the stringent requirements of the retina. We
anticipate that other factors might provide similar fine-tuning to Egfr and
Notch signaling in other tissues.
In the echinoid null allele described here
(edl(2)k01102), only 54% of ommatidia contain multiple R8s
(fewer by Boss staining), suggesting that another factor may be acting
redundantly to downregulate Egfr signaling in some cells. One candidate for a
redundant factor is a highly homologous gene distal to echinoid on
the second chromosome. Preliminary data indicates that this gene, which we
refer to as fred (friend of echinoid), is expressed in the
same tissues as echinoid and displays similar interactions with
EgfrEllipse (S.S., unpublished)
(The FlyBase Consortium,
2003). Further examination of the fred phenotype and
creation of fred ed lines will be necessary to determine if
fred acts in a manner similar to echinoid.
In its extracellular domain, Echinoid appears similar to other members of
the L1 family of proteins: it undergoes homophilic binding and ectodomain
shedding, presumably to regulate cell-cell adhesion. Although some L1 cell
adhesion proteins have been shown to interact with receptor tyrosine kinases
such as Egfr, those that have been described to date lead to activation, not
inhibition, of MAP kinase phosphorylation
(Schaefer et al., 1999;
Thelen et al., 2002
). In
addition, Echinoid lacks two intracellular motifs common to many L1 proteins:
a clathrin sorting motif (YRSLE), which regulates internalization, and an
ankyrin-binding domain (NEDGSFIGQY), which controls association with the
cytoskeleton, suggesting that Echinoid acts by a different mechanism from
other L1 proteins. As we find that overexpression of Echinoid in tissue has no
effect on the level of phosphorylated MAP kinase, a read-out of Egfr
signaling, it appears that Echinoid does not act as a general inhibitor of
Egfr. Instead, the prolonged presence of phosphorylated MAP kinase in
echinoid mutants suggests that the role of Echinoid is to
downregulate Egfr signaling after a period of activation. Below, we explore
possible (and not mutually exclusive) models for the function of Echinoid.
The ability of Egfr to signal depends on its localization and its
downstream targets. Ligand-induced endocytosis is a well-documented mechanism
for downregulating Egfr activity (for a review, see
Carpenter, 2000), and the
prolonged Egfr signaling we observe in echinoid mutants suggests that
one possible role for Echinoid is to facilitate Egfr endocytosis after a
period of activity. Another notable feature of Echinoid is its unusual
intracellular domain, which differs from other members of the L1 superfamily.
This domain is likely required for at least some aspects of Echinoid function
(Bai et al., 2001
) (see above),
and suggests that Echinoid may target downstream signaling molecules. Based on
our results, this unknown pathway would intersect with Egfr signaling prior to
MAPK phosphorylation.
What downstream molecules might be targeted by Echinoid? One potential
model for the function of Echinoid is provided by work on the vertebrate
SIRP proteins, the only group of Ig-containing proteins shown to
inhibit receptor-tyrosine kinase (RTK) signaling
(Kharitonenkov, 1997
)
(reviewed by Cant and Ullrich,
2001
; Vely and Vivier,
1997
). SIRP-
proteins are phosphorylated on tyrosine in
response to RTK activation; these phosphorylated residues provide binding
sites for the SHP2 tyrosine phosphatase. Analysis of the Drosophila
genomic sequence uncovered no clear Drosophila orthologs of
SIRP-
proteins, but the overall structural similarity of Echinoid, its
phosphorylation in response to dEGFR signaling and its importance in
downregulating dEGFR signaling suggest that it may function in a manner
analogous to the SIRP-
proteins. We have observed genetic interactions
between echinoid and corkscrew, the Drosophila
homolog of SHP2, and have been able to detect binding between these proteins
in cultured cells (S.S., unpublished). However, the significance of these
interactions will require further study in vivo.
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ACKNOWLEDGMENTS |
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