1 Department of Cell and Developmental Biology, University of Michigan, Medical
School, Ann Arbor, MI 48109-0616, USA
2 Institute of Molecular Medicine, Department of Life Science, National Tsing
Hua University, Hsinchu, Taiwan 30043, Republic of China
Authors for correspondence (e-mail:
hortsch{at}umich.edu
and
lshsu{at}life.nthu.edu.tw)
Accepted 23 January 2003
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: EGF receptor, Cell adhesion, Echinoid, Neuroglian, Signaling, Drosophila
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Drosophila eye is composed of about 800 ommatidia, each of
which includes eight light-sensing photoreceptor neurons (R1-R8), four
non-neuronal cone cells and eight accessory cells. EGFR activity is required
for the differentiation of all cell types, with the exception of photoreceptor
R8 cells (Dominguez et al.,
1998; Freeman,
1996
). In this study, we have used the development of the
Drosophila compound eye as the experimental paradigm to explore the
physical and functional interactions between two Drosophila Ig-domain
containing CAMs, Echinoid (Ed) and Neuroglian (Nrg), and their effect on EGFR
signaling. The Ig superfamily is well represented in the Drosophila
genome, which contains about 150 genes encoding Ig domain proteins. Many of
these gene products have cell adhesion functions and fulfill important roles
during Drosophila development
(Hynes and Zhao, 2000
). Ed has
seven Ig domains, two fibronectin type III (Fn III) domains and a
transmembrane (TM) domain, followed by a 315 amino acid intracellular domain
with no identifiable structural or functional amino acid motif
(Bai et al., 2001
). ed
was originally isolated as an enhancer of the rough eye phenotype caused by
ElpB1, a gain-of-function Egfr allele, and
genetically interacts with several components in the EGFR pathway. As a
consequence, the ed mutant phenotype includes the generation of extra
photoreceptor and cone cells in the Drosophila eye. Conversely,
overexpression of ed in the eye leads to a reduction of photoreceptor
cell number. These results indicate that ed is a negative regulator
of the EGFR pathway. Based on genetic mosaic and epistatic analyses, we
proposed that Ed acts as a homotypic cell adhesion protein, which antagonizes
EGFR signaling by regulating the activity of the TTK88 transcriptional
repressor, the most downstream component of the EGFR signaling pathway
(Bai et al., 2001
).
L1-type proteins comprise six Ig domains, three to five Fn III repeats and
a cytoplasmic domain with a conserved ankyrin-binding site. This family of
transmembrane proteins includes L1-CAMs, neurofascins, NrCAMs, NgCAM and
CHL1s, in vertebrates, and Neuroglians, in invertebrate species (for a review,
see Brümmendorf et al.,
1998; Hortsch,
2000
). During nervous system development, L1-type CAMs have been
implicated in neurite outgrowth, axon guidance and neurite fasciculation,
which employ both homophilic and heterophilic interactions (for a review, see
Crossin and Krushel, 2000
;
Hortsch, 2000
). Mutations in
the human L1CAM gene result in mental retardation and other
neurological phenotypes, for which summarily the term CRASH syndrome has been
coined. This emphasizes the importance of L1CAM for the development
of the nervous system (Kamiguchi et al.,
1998
). Nrg is the Drosophila homolog of the vertebrate L1
family proteins (Bieber et al.,
1989
). Alternative splicing of the primary transcript produces two
protein isoforms of Nrg, which differ in their intracellular domain
(Hortsch et al., 1990
). The
Nrg180 isoform is neuron specific, whereas the Nrg167
isoform is expressed more broadly. The presence of a highly conserved FIGQY
ankyrin-binding site enables Nrg and other L1-type proteins to assemble
membrane skeleton components at sites of cell-cell contact
(Dubreuil et al., 1996
;
Hortsch et al., 1998
).
Phosphorylation of the tyrosine residue in the FIGQY motif abolishes this
ankyrin-binding activity (Garver et al.,
1997
; Tuvia et al.,
1997
). An analysis of Drosophila lines with homozygous
lethal mutations in the nrg gene demonstrated alterations in
motoneuron axon pathfinding and other neurological phenotypes
(Hall and Bieber, 1997
). In
addition, Nrg autonomously increases the activity of both the EGFR and FGFR to
control growth cone decisions
(Garcia-Alonso et al., 2000
)
(R.I. and M.H., unpublished). Therefore, both Ed and Nrg are involved in the
regulation of RTK signaling processes.
In this paper, we have used a genetic co-expression screen to identify Nrg as a non-autonomous ligand of Ed. When compared with overexpression of either ed or nrg alone, co-expression of both molecules together uncovers a specific synergistic effect in inhibiting EGFR signaling. By using a S2 cell expression system we also demonstrate a direct heterophilic trans-interaction between Ed and Nrg. The observation that only the intracellular domain of Ed is required for the EGFR signal repression leads us to propose that Ed is the signal-receiving molecule and is activated by either its own homophilic interaction or by Nrg. Subsequently, it autonomously represses the EGFR signaling pathway by a so far unknown mechanism.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Molecular biology
UAS-edC50 was generated by ligating two overlapping PCR
products, with the first PCR product containing the TM domain plus the
following 22 amino acids of Ed and a second PCR product containing the last
C-terminal 50 amino acids of Ed. For the S2 cell expression studies, cDNAs
were subcloned into the pRmHa3 vector under the control of the
Drosophila metallothioneine promoter
(Bunch et al., 1988). The
constructs for Drosophila Nrg180and NrgGPI have
been described previously (Hortsch et al.,
1995
). The pRmHa3-Drosophila Fasciclin 1 plasmid was
constructed by ligating a 3.0 kb EcoRI Fasciclin 1 cDNA fragment into
the EcoRI restriction site of the pRmHa3 vector. The Echinoid cDNA
was subcloned into pRmHa3 as a 4.3 kb SmaI/SalI fragment and
subsequently modified by the removal of a 3' 0.5 kb
NsiI/SalI fragment, which was replaced by an oligonucleotide
cassette encoding the HA-epitope
(Kolodziej and Young, 1991
).
This substitutes the 73 C-terminal amino acid residues of the natural Ed
protein with two copies of the HA-epitope (YPYDVPDYA).
Histology
Scanning electron micrographs were prepared as described
(Kimmel et al., 1990).
Immunohistochemical staining of imaginal discs was performed as described
(Bai et al., 2001
)using
anti-ELAV (rat, 1:250, Developmental Studies Hybridoma Bank), anti-Cut (mouse,
1:5, Developmental Studies Hybridoma Bank), anti-Ed (rabbit, 1:200)
(Bai et al., 2001
) and 1B7
(mouse, 1:200) (Bieber et al.,
1989
). Cy3- and Cy5-conjugated secondary IgGs are from Jackson
ImmunoResearch Laboratories. Confocal microscopy was performed using Zeiss
Model Pascal.
Transfection and maintenance of S2 cells
Schneider 2 (S2) cells were maintained at 25°C in Schneider's medium
with 10% fetal calf serum and penicillin/streptomycin (all reagents were from
Life Technologies). S2 cells were transfected with Lipofectin Reagent (Life
Technologies) according to the manufacturer's protocol and transfected cells
were selected using hygromycin resistance (250 µg/ml hygromycin B; Sigma),
which was conferred by the pCOhygro plasmid (Invitrogen). A
detailed protocol for establishing cloned S2 cell lines using soft agar
cloning has been reported previously
(Bieber, 1994). Individual cell
clones were induced overnight with 0.7 mM CuSO4 and analyzed by
Western blotting for high expression of the transfected cDNAs. Selected lines,
designated S2:Ed, S2:Nrg180, S2:NrgGPI or S2:Fas1,
expressed either the HA-epitope-tagged form of Drosophila Echinoid,
the neuronal or the artificial GPI-anchored isoform of Drosophila
Neuroglian or Drosophila Fasciclin 1, respectively.
Cell aggregation assays
Usually 2x106 cells were labeled using the
Cell-TrackerTM CM-DiI reagent (Molecular Probes) for 2 hours at 25°C
in serum-free medium. The labeled cells were washed with complete S2 cell
medium five times to remove excess dye and induced overnight with 0.7 mM
CuSO4. Labeled, induced cells were mixed with unlabeled, induced
cells as indicated to a final concentration of 1.5x106
cells/ml and incubated on a rotating shaker at 200 rpm for 2 hours at room
temperature. Small aliquots of aggregated cells were mounted on microscope
slides under a #2 coverslip bridge and examined and photographed using a Nikon
Optiphot 2 microscope (Nikon), which was equipped with Nomarski and rhodamine
channel epifluorescence optics and a Nikon DXM1200 digital camera.
SDS-polyacrylamide gel electrophoresis and western blot analysis
Induced S2 cells were collected by centrifugation (2.5x105
cells/lane) and solubilized using SDS-polyacrylamide sample buffer.
Solubilized S2 cell proteins or immunoprecipitated proteins were separated by
electrophoresis in 10% SDS-polyacrylamide gels and transferred onto
nitrocellulose filters. Subsequently, the blots were probed with specific
primary and with horseradish peroxidase-conjugated secondary antibodies
(Jackson ImmunoResearch Laboratories) and developed with
3,3'-diaminobenzidine as described by Hortsch et al.
(Hortsch et al., 1985). The
HA.11 monoclonal antibody was a gift from Dr R. Dubreuil (University of
Illinois at Chicago). The 1B7, BP-104 and the 3C1 mouse monoclonal antibodies
against Drosophila Neuroglian have been described and characterized
previously (Bieber et al.,
1989
; Hortsch et al.,
1990
; Hortsch et al.,
1995
).
Co-immunoprecipitation procedure
Immunoprecipitations were performed using a modification of the protocol by
Anderson and Blobel (Anderson and Blobel,
1983). Transfected cells were induced overnight with 0.7 mM
CuSO4 and mixed and incubated on a shaking platform as indicated. A
total of 10x106 cells were collected by centrifugation for
each immunoprecipitation and solubilized in a buffer containing 1.25% Trition
X-100. The soluble fraction was incubated overnight at 4°C with either 1B7
anti-Nrg or with a control monoclonal antibody. 50 µl of a 1:1 Protein
G-Sepharose suspension (Amersham Pharmacia Biotech) was added and incubated at
room temperature on a rotator for 2 hours. The Protein G-Sepharose beads were
collected by centrifugation and washed four times. Bound proteins were eluted
with SDS-polyacrylamide gel electrophoresis sample buffer and probed after
SDS-polyacrylamide gel electrophoresis by western blot analysis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We used the GMR-GAL4 driver line to co-express UAS-ed with several
available UAS and EP lines that drive overexpression of various Ig
domain-containing adhesion molecules. As shown previously, ectopic expression
of Ed in the eye results in a rough eye phenotype and a loss of photoreceptor
and cone cells (Bai et al.,
2001) (Fig.
1B,F,J). On average, 10-15% of ommatidia were missing
photoreceptor or cone cells (Table
1). By contrast, overexpression of either the neuronal
nrg180 or the non-neuronal nrg167
isoform alone had no effect on the number of photoreceptor or cone cells
(Fig. 1C,G,K; Table 1). However,
co-expression of both ed and nrg180 (or
nrg167) resulted in a more severe rough eye phenotype
(Fig. 1D) with a reduction of
the number of ommatidia, a varying size of ommatidia and a decrease in the
number of bristles. In addition, a significantly higher percentage of
ommatidia contained fewer photoreceptor and cone cells
(Fig. 1H,L;
Table 1). No synergistic
effects were detected when ed was overexpressed together with other
CAMs, such as Drosophila Fasciclin 2 or human L1CAM (data not
shown).
|
|
In the developing Drosophila imaginal eye disc Ed is
colocalized with Nrg
The genetic synergy between Ed and Nrg suggests that both proteins might
also physically interact with each other. Using antibodies that specifically
recognize Ed and both isoforms of Nrg for an immunocytochemical analysis, we
first tested this possibility by examining their expression pattern in the
developing Drosophila eye disc. Both Ed and Nrg are colocalized to
all cells throughout the third instar larval eye disc, including
undifferentiated cells (Fig.
2A-C) and developing ommatidial clusters
(Fig. 2D-F).
|
|
|
|
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
The non-neuronal isoform of Nrg (Nrg167) is expressed in the
non-neuronal, epithelial cells of eye imaginal discs. It exhibits a similar
effect on Ed (and thereby the EGFR signaling pathway) as does the neuronal Nrg
isoform (Nrg180), which is expressed by the photoreceptor cells
(data not shown). Therefore, Nrg167 is probably the major Nrg
isoform that inhibits the intrinsic EGFR signaling for basally located,
undifferentiated cells. Although our S2 cell mixing experiments clearly show
that Ed and Nrg protein interact with each other in a trans-type modus, our
results neither prove nor disprove that they might also interact in a cis-type
modus. In fact, some Ig-domain CAMs, such as axonin 1/TAG1, interact with
L1-type proteins exclusively in a functional cis-type interaction
(Malhotra et al., 1998).
Nrg is an autonomous activator of RTK
Genetic evidence indicates that Nrg is a cell-autonomous, positive
regulator of EGFR signaling in neuronal cells that express both Nrg and EGFR
(Garcia-Alonso et al., 2000)
(R.I. and M.H., unpublished). However, in the developing Drosophila
eye disc Nrg functions non-autonomously as a ligand of Ed and activates Ed in
the neighboring cells to repress downstream EGFR signaling. Thus, depending on
the cellular context, Nrg can act both as an autonomous activator, as well as
a non-autonomous inhibitor of the EGFR signaling pathway.
Autonomous versus non-autonomous effects of ed on EGFR
signaling
Our previous genetic mosaic analysis indicated that ed acts in a
cell non-autonomous manner (Bai et al.,
2001). As the intracellular domain of Ed is required for EGFR
signal repression, we propose that through its homophilic interaction Ed
transmits a negative signal in the receiving cell and antagonizes the EGFR
pathway. In this study, we demonstrate a homophilic adhesive activity of Ed,
and we further show that ed also acts autonomously as a heterophilic
receptor of Nrg. Thus, Ed appears to influence EGFR signaling through both
homophilic (non-autonomous) and heterophilic (autonomous) interactions, but
the relative contribution derived from either interaction is unknown. Flies
that are mutant for ed have extra photoreceptor and cone cells. By
contrast, when shifting temperature-sensitive nrg3 larvae
to the restrictive temperature during the third instar larval stage, we did
observe wild-type number of Elav- and Cut-positive cells (data not shown).
Therefore, the Nrg-mediated heterophilic activity of Ed in repressing EGFR
signaling appears to be redundant with the homophilic activity of Ed.
Further studies are required to reveal the molecular mechanism by which ed inhibits the EGFR signaling pathway. Equally, with ed and nrg widely expressed in the developing Drosophila eye disc, it remains to be revealed how the two opposing effects of nrg on EGFR activity might contribute to a differential cellular segregation and the development of different ommatidial cell types.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Anderson, D. J. and Blobel, G. (1983). Immunoprecipitation of proteins from cell-free translations. Methods Enzymol. 96,111 -120.[Medline]
Bai, J., Chiu, W., Wang, J., Tzeng, T., Perrimon, N. and Hsu,
J. (2001). The cell adhesion molecule Echinoid defines a new
pathway that antagonizes the Drosophila EGF receptor signaling
pathway. Development
128,591
-601.
Bieber, A. J. (1994). Analysis of cellular adhesion in cultured cells. In Drosophila melanogaster: Practical Uses in Cell Biology, Vol. 44 (ed. L. Goldstein and E. Fyrberg), pp. 683-696. San Diego: Academic Press.
Bieber, A. J., Snow, P. M., Hortsch, M., Patel, N. H., Jacobs, J. R., Traquina, Z. R., Schilling, J. and Goodman, C. S. (1989). Drosophila neuroglian: a member of the immunoglobulin superfamily with extensive homology to the vertebrate neural adhesion molecule L1. Cell 59,447 -460.[Medline]
Brümmendorf, T., Kenwrick, S. and Rathjen, F. G. (1998). Neural cell recognition molecule L1: from cell biology to human hereditary brain malformations. Curr. Opin. Neurobiol. 8,87 -97.[CrossRef][Medline]
Bunch, T. A., Grinblat, Y. and Goldstein, L. S. B. (1988). Characterization and use of the Drosophila metallothionein promoter in cultured Drosophila melanogaster cells. Nucleic Acids Res. 16,1043 -1061.[Abstract]
Crossin, K. L. and Krushel, L. A. (2000). Cellular signaling by neural cell adhesion molecules of the immunoglobulin superfamily. Dev. Dyn. 218,260 -279.[CrossRef][Medline]
Dominguez, M., Wasserman, J. D. and Freeman, M. (1998). Multiple functions of the EGF receptor in Drosophila eye development. Curr. Biol. 8,1039 -1048.[Medline]
Dubreuil, R. R., MacVicar, G., Dissanayake, S., Liu, C., Homer, D. and Hortsch, M. (1996). Neuroglian-mediated cell adhesion induces assembly of the membrane skeleton at cell contact sites. J. Cell Biol. 133,647 -655.[Abstract]
Elkins, T., Hortsch, M., Bieber, A. J., Snow, P. M. and Goodman, C. S. (1990). Drosophila fasciclin I is a novel homophilic adhesion molecule that along with fasciclin III can mediate cell sorting. J. Cell Biol. 110,1825 -1832.[Abstract]
Freeman, M. (1996). Reiterative use of the EGF receptor triggers differentiation of all cell types in the Drosophila eye. Cell 87,651 -660.[Medline]
Garcia-Alonso, L., Romani, S. and Jimenez, F. (2000). The EGF and FGF receptors mediate neuroglian function to control growth cone decisions during sensory axon guidance in Drosophila.Neuron 28,741 -752.[Medline]
Garver, T. D., Ren, Q., Tuvia, S. and Bennett, V.
(1997). Tyrosine phosphorylation at a site highly conserved in
the L1 family of cell adhesion molecules abolishes ankyrin binding and
increases lateral mobility of neurofascin. J. Cell
Biol. 137,703
-714.
Hall, S. G. and Bieber, A. J. (1997). Mutations in the Drosophila neuroglian cell adhesion molecule affect motor neuron pathfinding and peripheral nervous system patterning. J. Neurobiol. 32,325 -340.[CrossRef][Medline]
Hortsch, M. (1996). The L1 family of neural cell adhesion molecules: old proteins performing new tricks. Neuron 17,587 -593.[Medline]
Hortsch, M. (2000). Structural and functional evolution of the L1-family: Are four adhesion molecules better than one? Mol. Cell. Neurosci. 15,1 -10.[CrossRef][Medline]
Hortsch, M., Avossa, D. and Meyer, D. I.
(1985). A structural and functional analysis of the docking
protein. Characterization of active domains by proteolysis and specific
antibodies. J. Biol. Chem.
260,9137
-9145.
Hortsch, M. and Bieber, A. J. (1991). Sticky molecules in not-so-sticky cells. Trends Biochem. Sci. 16,283 -287.[CrossRef][Medline]
Hortsch, M., Bieber, A. J., Patel, N. H. and Goodman, C. S. (1990). Differential splicing generates a nervous system-specific form of Drosophila neuroglian. Neuron 4, 697-709.[Medline]
Hortsch, M., O'Shea, K. S., Zhao, G., Kim, F., Vallejo, Y. and Dubreuil, R. R. (1998). A conserved role for L1 as a transmembrane link between neuronal adhesion and membrane cytoskeleton assembly. Cell Adhes. Commun. 5, 61-73.[Medline]
Hortsch, M., Wang, Y. M., Marikar, Y. and Bieber, A. J.
(1995). The cytoplasmic domain of the Drosophila cell
adhesion molecule neuroglian is not essential for its homophilic adhesive
properties in S2 cells. J. Biol. Chem.
270,18809
-18817.
Hynes, R.O. and Zhao, Q. (2000). The evolution of cell adhesion. J. Cell Biol. 150,F89 -F95.[Medline]
Kamiguchi, H., Hlavin, M. L., Yamasaki, M. and Lemmon, V. (1998). Adhesion molecules and inherited diseases of the human nervous system. Annu. Rev. Neurosci. 21, 97-125.[CrossRef][Medline]
Kimmel, B. E., Heberlein, U. and Rubin, G. M. (1990). The homeo domain protein rough is expressed in a subset of cells in the developing Drosophila eye where it can specify photoreceptor cell subtype. Genes Dev. 4, 712-727.[Abstract]
Kolodziej, P. A. and Young, R. A. (1991). Epitope tagging and protein surveillance. Methods Enzymol. 194,508 -519.[Medline]
Malhotra, J. D., Tsiotra, P., Karagogeos, D. and Hortsch, M.
(1998). Cis-activation of L1-mediated ankyrin recruitment by
TAG-1 homophilic cell adhesion. J. Biol. Chem.
273,33354
-33359.
Spradling, A. C. and Rubin, G. M. (1982). Transposition of cloned P elements into Drosophila germ line chromosomes. Science 218,341 -347.[Medline]
Tuvia, S., Garver, T. D. and Bennett, V.
(1997). The phosphorylation state of the FIGQY tyrosine of
neurofascin determines ankyrin-binding activity and patterns of cell
segregation. Proc. Natl. Acad. Sci. USA
94,12957
-12962.
Williams, E. J., Furness, J., Walsh, F. S. and Doherty, P. (1994). Activation of the FGF receptor underlies neurite outgrowth stimulated by L1, N-CAM, and N-cadherin. Neuron 13,583 -594.[Medline]