1 Department of Molecular Cell and Developmental Biology, University of California Los Angeles, Los Angeles, CA 90095, USA
2 Department of Zoology, University of Toronto, Toronto, Ontario M5S3G5, Canada
* These authors contributed equally to this work
Author for correspondence (e-mail: volkerh{at}mcdb.ucla.edu)
Accepted 22 May 2002
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SUMMARY |
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Key words: EGFR signaling, DE-cadherin, shotgun, Morphogenesis, Adhesion, Visual system, Drosophila melanogaster
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INTRODUCTION |
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In Drosophila and vertebrates, there are numerous examples where adhesion among epithelial cells has to be adjusted dynamically to allow for morphogenetic movements, such as ingression, invagination, or cell migration to occur. Here we study DE-cadherin function in the formation of the Drosophila optic placodes and their subsequent differentiation. Progenitors of the insect central nervous system, called neuroblasts, typically delaminate from the neurectoderm as individual cells, whereas neighboring cells thaat stay behind in the neurectoderm form epidermal progenitors. Some regions within the insect neurectoderm, among them the optic placode, develop similarly to the vertebrate neural tube: instead of forming neuroblasts by delamination, these regions form placodes of highly cylindrical cells that invaginate and form internal neuroepithelial vesicles in which cells maintain their apical-basal polarity and junctional complex (Dumstrei et al., 1998). The optic placode splits into two lineages, one that forms the larval eye (Bolwigs organ), and the other that gives rise to the optic lobe. Cells of Bolwigs organ differentiate as sensory neurons that send their axons to the optic lobe (Steller et al., 1987
; Green et al., 1993
).
Cadherins can be regulated on the level of transcription, which is often seen when transitions between epithelial and mesenchymal cells take place such as neural crest in vertebrates, neuroblast or heart precursors in Drosophila (Duband et al., 1995; Tepass et al., 1996
; Haag et al., 1999
). A more rapid mechanism of controlling cadherin activity is by modifying its coupling to the actin cytoskeleton. Tyrosine phosphorylation of ß-catenin may result in a disassembly of the CCC and a consecutive loss in cadherin-mediated adhesion (Aberle et al., 1996
; Ozawa and Kemler, 1998
; Provost and Rimm, 1999
). Several tyrosine kinases and tyrosine phosphatases have been identified that can increase or decrease the degree of phosphorylation of the CCC. In vertebrates, receptor tyrosine kinases, including EGF receptor, were shown to be physically linked to the CCC and to be responsible for CCC phosphorylation (Hoschuetzky et al., 1994
; Hazan and Norton, 1998
). We speculated that the Drosophila EGF receptor homolog, EGFR, may play a similar role in modulating DE-cadherin mediated adhesion during tissue morphogenesis. This aspect of DE-cadherin mediated adhesion had not previously been addressed. It is known that the cytoplasmic pool of the Drosophila ß-catenin homolog, Armadillo (Arm) is phosphorylated on serine/threonine residues as part of the Wingless pathway (see Peifer et al., 1994
); the localization and significance of phosphorylated tyrosine residues in Arm has not been studied.
In this paper we show that finely adjusted DE-cadherin-mediated adhesion is required for normal optic placode morphogenesis. In embryos that lack DE-cadherin, this structure dissociates and undergoes apoptotic cell death. Overexpression of DE-cadherin results in the failure of optic placode cells to invaginate, and of Bolwigs organ precursors to separate from the placode. This phenotype is also observed in embryos that lack the EGFR when the widespread cell death (the most prominent aspect of loss of EGFR) is suppressed, suggesting that compromising EGFR signaling results in an increased adhesion of optic placode cells. This notion is corroborated by the genetic interactions we find between DE-cadherin and EGFR signaling, and by the fact that EGFR forms part of the CCC as shown by co-immunopreciptation from embryonic extracts. These results suggest that EGFR signaling negatively regulates DE-cadherin activity, thereby facilitating the invagination of the optic placodes.
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MATERIALS AND METHODS |
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Immunohistochemistry
Embryos were dechorionated and fixed in 4% formaldehyde containing PT (1% PBS, 0.3% Triton X-100) with heptane. Embryos were then devitellinized in methanol and stored in ethanol prior to labeling with antibody, following the standard procedure (Ashburner, 1989). Embryos stained with anti-Armadillo antibody were heat fixed (Miller et al., 1989
). This method greatly emphasizes the signal at the adherens junction. Anti ß-galoctosidase antibody (Sigma) was used at 1:1000. A monoclonal antibody against Armadillo, which labels the zonula adherens junction (Peifer, 1993
) (provided by the Developmental Studies Hybridoma Bank), was used at 1:100. Anti-phosphohistone H3 antibody, which labels dividing cells, (available from Upstate Biotechnology) was used at 1:500. A monoclonal antibody against Crumbs protein that labels apical membranes of ectodermal tissues (Tepass et al., 1990
) (kindly provided by Dr E. Knust) was used at 1:20. Antibody against Coracle, which marks the septate junctions was used at 1:250 (Fehon et al., 1994
). Antibody against EGFR (Zak et al., 1990
) (kindly provided by Dr B. Shilo) was used at 1:200. A commercial monoclonal antibody against activated MAPK (dp-ERK) (available through Sigma), used at 1:200, was used to visualize the embryo domains in which the Ras signaling pathway is activated (Gabay et al., 1997a
; Gabay et al., 1997b
). A monoclonal antibody that recognizes the Fasciclin ll protein (Fasll) (Grenningloh et al., 1991
) (kindly provided by Dr C. Goodman) which labels subsets of neuronal precursors, among them part of the optic lobe, larval eye and dorsomedial brain was used at 1:100. Fasciclin lll antibody (Faslll) (Patel et al., 1987
) (provided by the Developmental Studies Hybridoma Bank) labels the basolateral surface of ectodermal tissue was used at 1:100. The monoclonal antibody mAb22C10 (Zirpursky et al., 1984
) (provided by the Developmental Studies Hybridoma Bank) which labels all neurons was used at 1:200. Anti DE-cadherin antibody was used at 1:20 (kindly provided by Dr T. Uemura) (Uemura et al., 1996
). Confocal images were taken on a Biorad MRC 1024ES miroscope using Biorad Lasersharp version 3.2 software.
In situ hybridization
Digoxigenin-labeled DNA probe was prepared following manufacturers instructions (Genius kit; Boehringer) using a full-length cDNA clone of the rpr gene (White et al., 1994) (kindly provided by Dr K. White). Embryos were dechorionated and fixed in PBS containing 5% formaldehyde and 50 mM EGTA and stored in ethanol. In situ hybridizations to whole-mount embryos were carried out according to the protocol of Tautz and Pfeifle (Tautz and Pfeifle, 1989
). Embryos were dehydrated in ethanol and embedded in epon.
Temperature shift experiments
Egfrf1 embryos were collected for 2 hours at 25°C, and shifted to 31°C at 3, 4, 5 and 6 hours post-fertilization. Embryos remained at 31°C for 2 hours and were then allowed to develop at 22°C until stage 16 of embryogenesis, at which time they were fixed for subsequent staining. Completion of embryogenesis takes 42 hours at 18°C, 16 hours at 29°C, and 22 hours at 25°C. To compensate for timing differentials, ratios of these hours were used.
Scanning electron microsocopy (SEM)
Wild-type and Egfrf5 embryos were fixed and devitellinized using the same protocol as for immunohistochemistry. They were then dehydrated in ethanol and washed twice for 10 minutes in Hexamethyldisilazane (available from Ted Pella Inc.). SEM images were made on a Hitachi model # S-2460N at 15 kV. To identify Egfrf5 homozygous mutants a second chromosome balancer containing a P element with a ftz-lacZ promoter fusion construct was used. The Egfrf5 line was stained with anti ß-galoctosidase antibody and homozygous mutant embryos were selected and processed as stated above.
Immunoprecipitation and immunoblotting
0- to 15-hour old embryos were collected and dechorianted with bleach for 3 minutes. 0.1 ml of embryo were homogenized with 0.2 ml of lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP40, 0.5% sodium deoxycholate, 1 mM DTT, 1 mM PMSF). Lysate was centrifuged at 13,000 g at 4°C for 10 minutes. The supernatant was pre-absorbed with 50 µl protein A-agarose beads (Amersham Corp) at 4°C for 30 minutes with gentle agitation to eliminate non-specific binding of the proteins to the beads. Protein A-agarose beads were separated from the lysate by centrifugation for 1 minute at 13,000 g at 4°C. Anti-DE-cadherin (1:100) or anti-Armadillo/ß-catenin (1:50) or anti-EGFR (1:50) was incubated with the lysate for 1 hour at 4°C with gentle agitation. 25 µl of protein A-agarose beads were subsequently added to each lysate/antibody mixture and incubated over night at 4°C with gentle agitation. The immune complex was pelleted by centrifugation at 3,000 g at 4°C for 1 minute. The complex was washed 2 times with low stringency (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% NP40) and high stringency (50 mM Tris-HCl, pH 7.5, 0.1% NP40) buffers. The proteins were eluted by boiling the beads with 20 µl SDS-PAGE buffer for 10 minutes.
Eluted proteins were separated on 8% SDS-PAGE gel and electroblotted to PDF membrane (Bio-Rad). The blots were blocked with PBS containing 5% non fat milk and 0.3% Tween 20 (Sigma). The membranes were incubated with anti-DE-cadherin, anti-Armadillo, anti-EGFR and anti-BP106 (Patel et. al., 1987) (provided by the Developmental Studies Hybridoma Bank) antibodies at 1:1000, 1:500 1:200 and 1:300 dilution followed by peroxidase-conjugated antibody at 1:2000 dilution. After washing with buffer containing PBS and Tween 20, protein bands were visualized with ECL detection kit (Amersham Corp). Immunoprecipitation experiments were independently performed three times to confirm the results.
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RESULTS |
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A second type of junction, called a septate junction, develops in Drosophila epithelial cells at a slightly later stage than the zonula adherens (Tepass and Hartenstein, 1994). Septate junctions have been implicated in maintaining epithelial stability (Woods et al., 1997
). The Coracle protein forms part of the septate junctional complex (Fehon et al., 1994
), and an antibody against Coracle serves as a sensitive marker for this junction. Applying this marker to embryos of different stages we found that all ectodermally derived epithelia express Coracle, except for the optic lobe and the invaginations that form the stomatogastric nervous system (Fig. 2H). Accordingly, no septate junctions have been reported in previous electron microscopic investigations of these tissues (Green et al., 1993
; Tepass and Hartenstein, 1994
). The reliance on adherens junctions alone may make the optic lobe (and stomatogastric nervous system, not considered here) more susceptible to changes in the stability of these junctions, as those described below resulting from manipulations of DE-cadherin and EGFR.
Loss and gain of DE-cadherin function alter visual system morphogenesis
Both loss and overexpression of DE-cadherin affect the maintenance and morphogenesis of the visual system. Using antibody markers against neural and epidermal cells, we find a loss of epithelial integrity and an increase in cell death in the head epidermis, resulting in a partial or full exposure of the brain in late shg mutant embryos (Fig. 3B,D). Similar defects are seen in the optic placodes and the invagination, which in wild-type moves this placode inside the embryo, fails to take place, leaving cells at the outer surface. The defects in visual system morphogenesis in shg mutant embryos are probably due to the loss of epithelial tissue structure and increased apoptosis.
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Armadillo acts in cell adhesion and not Wg signaling in the embryonic development of the visual system
Armadillo (Arm), the Drosophila homolog of ß-catenin, is an effector of Wingless (Wg) signaling and a core component of the CCC (reviewed by Tepass, 1999; Peifer and Polakis, 2000
). A decrease or increase in the levels of DE-cadherin are capable of modulating Wg signaling through altering the levels of cytoplasmic Armadillo (Orsulic and Peifer, 1996
; Sanson et al., 1996
). Moreover, embryos that lack Wg display severe defects in head morphogenesis that also affect the visual system (data not shown). Thus, loss or gain of DE-cadherin could result in visual system defects not only by changing cell adhesion but also by changing Wg signaling activity. To differentiate between these two effects we analyzed embryos that lack Arm function in cell adhesion but not Wg signaling.
Embryos that do not express arm zygotically display a strong Wg loss-of-function phenotype, rather than dramatic defects in epithelial integrity that is typical for shg mutants. This led to the suggestion that maternally provided Arm is sufficient for the adhesion function of this molecule (Cox et al., 1996). We investigated the arm mutant defects in the embryonic visual system more closely in arm mutant embryos that express the arms14 construct, which is active in Wg signaling but not adhesion (Orsulic and Peifer, 1996
). Such embryos show no wg phenotype in the cuticle. However, they exhibited defects in the ventral epidermis and the head that resembled a weak to moderate shg mutant phenotype. In particular, there were foci of cell death in and around the optic placodes. This structure initially formed, but lost its epithelial structure and failed to invaginate in late embryos (Fig. 4B,C). Furthermore, we overexpressed a fusion protein in which the cytoplasmic tail of DE-cadherin was replaced by a truncated
-catenin protein (DE-cad-
-catenin) lacking the N-terminal domain. As this construct lacks Arm binding sites it does not titer out cytoplasmic Arm to block Wg signaling. da-Gal4; UAS-DE-cad-
-catenin embryos display similar visual system defects as da-Gal4; UAS-DE-cadherin5,9 embryos (Fig. 4D). Expression of DE-cad-
-catenin in a shg null background partially rescues the shg loss-of-function phenotype to the same extent that a full lenght DE-cadherin construct rescues the shg loss-of-function phenotype (data not shown). This shows that the fusion construct is able to interact normally with the actin cytoskeleton to support cell adhesion.These results indicate that the visual system phenotypes in shg and arm mutant embryos and those resulting from DE-cadherin overexpression are caused by preventing dynamic changes in cell adhesion needed during visual system morphogenesis.
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The early occurring cell death in the neurectoderm prevents other aspects of EGFR function being evaluated. To investigate the Egfr phenotype in the absence of cell death we studied Egfr mutant embryos that also were homozygous for Df(3L)H99, which uncovers several genes required for cell death (White et al., 1994). In double mutants, all cells of the head epidermis and optic lobe placode are preserved and express a normal array of markers. These markers include the regulatory genes tailless (tll) and orthodenticle (otd/oc, ocelliless), both widely expressed in the procephalic neurectoderm (Hirth et al., 1995
; Younossi-Hartenstein et al., 1997
), as well as antibodies for structural proteins such as Crumbs (apical cell membranes) (Tepass et al., 1990
), FasIII (basolateral cell membranes) (Patel et al., 1987
), 22C10 (Zipursky et al., 1984
), and Armadillo (zonula adherens) (Peifer, 1993
). The most conspicuous aspect of the Egfr;Df(3L)H99 phenotype is the optic lobe defect (Fig. 6A,B). Invagination of the placode does not take place, leaving it exposed at the surface of the embryo. Furthermore, the cells that would normally separate from the placode and become larval photoreceptors, the Bolwigs organ, remain part of the placode. Although they display a typical epithelial phenotype and are structurally indistinguishable from the surrounding optic lobe cells, these cells express specific neuronal markers such as 22C10 (Fig. 6C). In some cases, outgrowth of short, axon-like processes can be observed. The optic lobe placode defects resulting from loss of EGFR function resembles in every aspect the defects caused by overexpression of DE-cadherin.
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Genetic interactions between components of the EGFR signaling pathway and DE-cadherin
The similar character of the EGFR loss-of-function and the DE-cadherin gain-of-function phenotypes suggest that EGFR signaling may negatively regulate DE-cadherin activity. To test this hypothesis, we looked for genetic interactions between EGFR signaling and DE-cadherin. As all available shg mutant alleles have severe head defects, that include loss of optic lobes, we utilized another phenotype of the shg mutant embryos namely the cuticle defect that results from the loss of epithelial integrity of epidermal cells, as a genetic assay (Tepass et al., 1996; Uemura et al., 1996
). Embryos mutant for the hypomorphic allele shgP34-1, typically have minor defects in their ventral cuticle (Fig. 7B). A small fraction of these embryos have a more severe phenotype resembling that caused by a shg null allele, where most of the ventral cuticle is missing.
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EGFR forms part of the CCC in Drosophila embryo
In vertebrates, there is direct evidence that EGFR binds to the CCC. Upon EGF stimulation, activated EGFR phosphorylates ß-catenin resulting in dissociation of Armadillo from E-cadherin, thus weakening cell adhesion (Hazan and Norton, 1998).
Results reported above show that EGFR and DE-cadherin are expressed in an overlapping pattern and interact genetically in Drosophila embryo. To establish a direct link between EGFR and the CCC, proteins from 0- to 15-hour old embryos were extracted, and immunoprecipitation (IP) using antibodies against DE-cadherin, Armadillo and EGFR were preformed. The same antibodies were used to probe western blots, which contained the IP products. The IP results indicate that EGFR forms a complex with the CCC, consistent with our genetic data. EGFR is detected in both anti-DE-cadherin and anti-Armadillo IP products (Fig. 8A,B). Inversely, IP using anti-EGFR antibody confirms the interaction between EGFR and CCC (Fig. 8C). To verify the specificity of the EGFR-CCC interaction, the membrane protein neurotactin (Patel et al., 1987) was used on western blots containing anti-DE-cadherin and anti-Armadillo IP products. As indicated in Fig. 8D, neurotactin does not associate with the CCC. We obtained the same result with numerous other membrane and secreted proteins, including the Wingless protein (not shown).
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DISCUSSION |
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Overexpression of DE-cadherin or the DE-cad--catenin fusion construct causes a dramatic change in optic lobe morphogenesis, without causing much disruption in other epithelia (K. D., unpublished). We speculate that this enhanced sensitivity of optic lobe cells towards an increased level of DE-cadherin may be in part due to the fact that adherens junctions form the only means of contact between optic lobe cells. In other epithelia, such as epidermis, trachaeae and hindgut, septate junctions form by far the more prominent junctional complex. Septate junctions have been implicated in epithelial stability from a number of genetic studies (Woods et al., 1997
) (reviewed by Tepass et al., 2001
). One could surmise that embryonic epithelia, as they enter the phase of differentiation during mid-embryogenesis, construct septate junctions that add to the adherens junctions developed at an earlier stage. This additional junctional complex makes late epithelia more resistant to changes in cadherins, a notion supported by the finding that blocking cadherins (by applying calcium chelators, or tyrosine kinase inhibitors) in early embryos up to stage 10 leads to a break down of epithelia, whereas it has only a small effect in later stages (F. W., unpublished). The optic lobe, which does not differentiate but gives rise to a population of neuroblasts later dring the larval period, does not form septate junctions, which could account for its strong reliance on normally functioning adherens junctions.
Expression of a fusion construct, DE-cad--catenin, in which the cytoplasmic domain of DE-cadherin is replaced by a truncated
-catenin, thereby preventing a reduction in the cytoplasmic pool of Arm, results in a similar phenotype as overexpressing full length DE-cadherin. This finding lends support to the notion that dissociation of the CCC may not occur at the interface between DE-cadherin and Arm or Arm and
-catenin. If one were to assume that dissociation occurred between any components of the CCC, one would expect a stronger phenotype, given that by overexpressing the fusion construct one not only increases the amount of DE-cadherin molecules interconnecting cells, but also the stability with which they are coupled to the cytoskeleton. Biochemical studies in vertebrates (Ozawa et al., 1998
; Takahashi et al., 1997
; Tsukatani et al., 1997
) and our own analysis (F. W. and V. H., unpublished) also show that phosphorylation of the CCC does not result in increased dissociation of Arm or
-catenin from the CCC, suggesting that the dissociation occurs distal of
-catenin.
The strength of the CCC and other structural molecules driving morphogenesis has to be controlled in a complex spatiotemporal pattern. Numerous widely conserved signaling pathways have been implicated in this process. In vertebrate embryos, mutations of the Wnt, Shh and BMP signaling pathways result in impressive examples which tissues and organs show defects in morphogenesis (Chiang et al., 1996; Furuta et al., 1997
; Goodrich and Scott, 1998
). Furthermore, it became clear that frequently signaling proteins affect fundamental cellular behaviors, such as proliferation, motility, adhesiveness and survival. This prompted the hypothesis that in many developmental scenarios, the proximal effect of receiving a signal could be a change in morphogenetic behavior (Moon et al., 1993a
; Moon et al., 1993b
; Ungar et al., 1995
; Ainsworth et al., 2000
; Chuong et al., 2000
). The discovery that one of the Wnt signal transducers, ß-catenin, leads a double life as a structural component of the cadherin-catenin complex, fueled the idea that Wnt signal could directly exert an effect on the adhesiveness on the cell, an idea that is supported by cell culture experiments (Bradly et al., 1993
; Hinck et al., 1994
). However, genetic studies demonstrated that in Drosophila, the roles of ß-catenin as a signaling transducer and a CCC component seem to be quite separate. Although it is clear that the cytoplasmic and membrane bound ß-catenin pools are in a steady state, binding of more ß-catenin to the membrane, by overexpression of DE-cadherin, reduces the cytoplasmic pool resulting in a wg minus phenotype (Sanson et al., 1996
). However, Wnt/Wg signaling seems to have no effect on the amount of membrane bound ß-catenin (Peifer et al., 1994
). Thus, in Drosophila, it appears that DE-cadherin mediated adhesion, at least under experimental conditions, interferes with Wnt/Wg signaling by competing for ß-catenin but Wnt/Wg signaling may not have a direct effect on adhesion mediated by the CCC.
Our findings suggest that another signaling pathway, the EGFR pathway, is involved in modulating cadherin-mediated adhesion and thereby controlling morphogenesis. In a previous paper (Daniel et al., 1999), we showed that EGFR, similar to its function in the developing compound eye, is activated in the precursors of the larval eye and adjacent optic lobe at a stage preceding optic lobe invagination and larval eye separation. The ligand for EGFR is Spitz, which is activated by Rhomboid.
In a small subset of larval eye precursors (the Bolwigs organ founders). As shown by Dumstrei et al. (Dumstrei et al., 1998), Daniel et al. (Daniel et al., 1999
) and in the present paper, loss of EGFR signaling results primarily in cell death, lending further support to the view that EGFR signaling functions generally in the ectoderm and its derivatives to maintain cell viability. Recent studies in Drosophila indicate that MAPK directly controls the expression and protein stability of the cell death regulator, Hid (W; Wrinkled) (Kurada and White, 1998
; Bergmann et al., 1998
). If cell death is prohibited by a deficiency of the reaper-complex, cells of the optic placode and most other embryonic cells that undergo apoptosis in EGFR loss of function survive. Both optic lobe and Bolwigs organ express several of their normal differentiation markers, but show a characteristic hyperadhesive phenotype, consisting in the failure of optic Iobe invagination and Bolwigs organ separation. Based on the similarity of this phenotype to that one resulting from DE-cadherin overexpression, and the genetic interaction between Egfr and DE-cadherin mutants in the ventral ectoderm, we propose that EGFR activation is required in normal development to phosphorylate the CCC and thereby allows optic lobe invagination and Bolwigs organ separation to occur. This would be in line with experimental results obtained in vertebrate cell culture studies, which demonstrated that drug- or EGFR-induced phosphorylation of the CCC leads to dissociation between CCC and cytoskeleton. Recent findings have shown that another phosphorylation event, mediated by the rho/rac GTPases, also effects adhesion by dissociating
-catenin from the CCC (Kaibuchi et al., 1999
).
Co-IP data indicates that EGFR is linked to the CCC in Drosophila as well. This implies that the effect of EGFR on DE-cadherin mediated adhesion could be a direct one that occurs at the cell membrane and does not involve MAPK signal transduction to the nucleus. It has been shown in a number of vertebrates cell culture systems that tyrosine phosphorylation of ß-catenin results in a disassembly of the CCC complex and a consecutive loss in cadherin-mediated adhesion (Aberle et al., 1996; Hazan and Norton, 1998
). Phenotypically, this results, among others, in increased invasiveness of tumor cell lines (Behrens et al., 1993
; Birchmeier, 1995
; Noë et al., 1999
), neuronal and growth cone motility (Lanier et al., 2000
), or inhibition of blastomere compaction (Ohsugi et al., 1999
). Several tyrosine kinases and phosphatases have been identified that can increase or decrease the degree of phosphorylation of the CCC. For example, v-src transfected into cultured cells phosphorylates ß-catenin and causes cells to dissociate, round up, and become more motile (Behrens et al., 1993
). EGFR also phosphorylates the CCC and forms an integral part of this complex (Hoschuetzky et al., 1994
). This opens up the possibility that growth factors, with their widespread expression and biological activity in the developing embryo, may exert part of their effect on cell behavior by modulating, in a rather direct way, cell adhesion at the membrane. Such a mechanism would account for the rapid mode of control of adhesion molecules. Systems such as the optic placode of the Drosophila embryo, where matters of different cell fates are decided at the same time when morphogenetic movements change the arrangement and shape of the cells involved, constitute favorable paradigms to address how signaling systems control both processes.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Aberle, H., Schwartz, H. and Kemler, R. (1996). Cadherin-catenin complex: protein interactions and their implications for cadherin function. J. Cell. Biochem. 61, 514-523.[Medline]
Ainsworth, C., Wan, S. and Skaer, H. (2000). Coordinating cell fate and morphogenesis in Drosophila renal tubules. Phil. Trans. Roy.Soc. Lond. B: Biol. Sci. 355, 931-937.
Ashburner, M. (1989). Drosophila. A Laboratory Manual. Cold Spring Harbor Press.
Behrens, J., Vakaet, L., Friis, R., Winterhager, E., van Roy, F., Mareel, M. M. and Birchmeier, W. (1993). Loss of epithelial differentiation and gain of invasiveness correlates with tyrosine phosphorylation of the E-cadherin/beta-catenin complex in cells transformed with a temperature sensitive v-SRC gene. J. Cell Biol. 120, 757-766.[Abstract]
Bergmann, A., Agapite, J., McCall, K. and Steller, H. (1998). The Drosophila gene hid is a direct molecular target of Ras-dependent survival signaling. Cell, 95, 331-341.[Medline]
Birchmeier, W. (1995). E-cadherin as a tumor (invasion) suppressor gene. BioEssays 17, 97-99.[Medline]
Bradley, R. S., Cowin, P. and Brown, A. M. (1993). Expression of Wnt-1 in PC12 cells results in modulation of plakoglobin and E-cadherin and increased cellular adhesion. J. Cell Biol. 123, 1857-1865.[Abstract]
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401-415.
Campos-Ortega, J. A. and Hartenstein, V. (1997). The Embryonic Development of Drosophila melanogaster. 2nd edn, pp. 233-257. Berlin: Springer-Verlag.
Chang, T., Mazotta, J., Dumstrei, K., Dumitrescu, A. and Hartenstein, V. (2001). Dpp and Hh signaling in the Drosophila embryonic eye field. Development 128, 4691-4704.
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383, 407-413.[Medline]
Chuong, C. M., Patel, N., Lin, J., Jung, H. S. and Widelitz, R. B. (2000). Sonic hedgehog signaling pathway in vertebrate epithelial appendage morphogenesis: perspectives in development and evolution. Cell. Mol. Life Sci. 57, 1672-1681.[Medline]
Cox, R. T., Kirkpatrick, C. and Peifer, M. (1996). Armadillo is required for adherens junction assembly, cell polarity, and morphogenesis during Drosophila embryogenesis. J. Cell Biol. 134, 133-148.[Abstract]
Daniel, A., Dumstrei, K., Lengyel, J. A. and Hartenstein, V. (1999). The control of cell fate in the embryonic visual system by atonal, tailless and EGFR signaling. Development 126, 2945-2954.
Duband, J. L., Monier, F., Delannet, M. and Newgreen, D. (1995). Epithelium-mesenchyme transition during neural crest development. Acta Anat. 154, 63-78.[Medline]
Dumstrei, K., Nassif, C., Abboud, G., Aryai, A., Aryai, A. R. and Hartenstein, V. (1998). EGFR signaling is required for the differentation and maintenance of neural progenitors along the dorsal midline of the Drosophila embryonic head. Development 125, 3417-3426.
Fehon, R. G., Dawson, I. A. and Artavanis-Tsakonas, S. (1994). A Drosophila homologue of membrane-skeleton protein 4.1 is associated with septate junctions and is encoded by the coracle gene. Development 120, 545-557.
Furuta, Y., Piston, D. W. and Hogan, B. L. M. (1997). Bone morphogenetic proteins (BMPs) as regulators of dorsal forebrain development. Development 124, 2203-2212.
Gabay, L., Seger, R. and Shilo, B. Z. (1997a). In situ activation pattern of Drosophila EGF receptor pathway during development. Science 277, 1103-1106.
Gabay, L., Seger, R. and Shilo, B. Z. (1997b). MAP kinase in situ activation atlas during Drosophila embryogenesis. Development 124, 3535-3541.
Godt, D. and Tepass, U. (1998). Drosophila oocyte localization is mediated by differential cadherin-based adhesion. Nature 395, 387-391.[Medline]
Goodrich, L. V. and Scott, M. P. (1998). Hedgehog and Patched in neural development and disease. Neuron 21, 1243-1257.[Medline]
Green, P., Hartenstein, A. Y. and Hartenstein, V. (1993). The embryonic development of the Drosophila visual system. Cell and Tiss. Res. 273, 583-598.
Grenningloh, G., Rehm, E. J. and Goodman, C. S. (1991). Genetic analysis of growth cone guidance in Drosophila Fasciclin II functions as a neuronal recognition molecule. Cell 67, 45-57.[Medline]
Haag, T. A., Haag, N. P., Lekven, A. C. and Hartenstein, V. (1999). The role of cell adhesion molecules in Drosophila heart morphogenesis: faint sausage, shotgun/DE-cadherin, and laminin A are required for discrete stages in heart development. Dev. Biol. 208, 56-69.[Medline]
Hazan, R. B. and Norton, L. (1998). The epidermal growth factor receptor modulates the interaction of E-cadherin with the actin cytoskeleton. J. Biol. Chem. 273, 9078-9084.
Hinck, L., Nelson, W. J. and Papkoff, J. (1994). Wnt-1 modulates cell-cell adhesion in mammalian cells by stabilizing beta-Catenin binding to the cell adhesion protein cadherin. J. Cell Biol. 124, 729-741.[Abstract]
Hirth, F., Therianos, S., Loop, T., Gehring, W. J., Reichert, H. and Furukubo-Tokunaga, K. (1995). Developmental defects in brain segmentation caused by mutations of the homeobox genes orthodenticle and empty spiracles in Drosophila. Neuron 15, 769-778.[Medline]
Holmes, A. L. and Heilig, J. S. (1999). Fasciclin II and Beaten path modulate intercellular adhesion in Drosophila larval visual organ development. Development, 126, 261-272.
Hoschuetzky, H., Aberle, H. and Kemler, R. (1994). Beta-Catenin mediates the interaction of the cadherin-catenin complex with epidermal growth factor receptor. J. Cell Biol. 127, 1375-1380.[Abstract]
Iwai, Y., Usui, T., Hirano, S., Steward, R., Takeichi, M. and Uemura, T. (1997). Axon patterning requires DN-cadherin, a novel neuronal adhesion receptor, in the Drosophila embryonic CNS. Neuron 19, 77-89.[Medline]
Kaibuchi, K., Kuroda, S., Fukata, M. and Nakagawa, M. (1999). Regulation of cadherin-mediated cell-cell adhesion by the Rho family GTPases. Curr. Opin. Cell Biol. 11, 591-596.[Medline]
Kurada, P. and White, K. (1998). Ras promotes cell survival in Drosophila by downregulating hid expression. Cell 95, 319-329.[Medline]
Lanier, L. M. and Gertler, F. B. (2000). From Abl to actin: Abl tyrosine kinase and associated proteins in growth cone motility. Curr. Opin. Neurobiol. 10, 80-87.[Medline]
Lindsley, D. L. and Zimm, G. G. (1992). The Genome of Drosophila melanogaster. San Diego: Academic Press Inc.
Lu, Y. and Settleman, J. (1999). The role of rho family GTPases in development: lessons from Drosophila melanogaster. Mol. Cell. Biol. Res. Commun. 1, 87-94.[Medline]
Miller, K. G., Field, C. M. and Alberts, B. M. (1989). Actin-Binding proteins from Drosophila embryos: A complex network of interacting proteins detected by F-actin affinity chromatography. J. Cell Biol. 109, 2963-2975.[Abstract]
Moon, R. T., DeMarais, A. and Olson, D. J. (1993a). Responses to Wnt signals in vertebrate embryos may involve changes in cell adhesion and cell movement. J. Cell Sci. Supplement 17, 183-188.
Moon, R. T., Campbell, R. M., Christian, J. L., McGrew, L. L., Shih, J. and Fraser, S. (1993b). Xwnt-5A: a maternal Wnt that affects morphogenetic movements after overexpression in embryos of Xenopus laevis. Development 119, 97-111.
Nassif, C., Daniel, A., Lengyel, J. A. and Hartenstein, V. (1998). The role of morphogenetic cell death during embryonic head development of Drosophila. Dev. Biol. 197, 170-186.[Medline]
Noë, V., Chastre, E., Bruyneel, E., Gespach, C. and Mareel, M. (1999) Extracellular regulation of cancer invasion: the E-cadherin-catenin and other pathways. Biochem. Soc. Symp. 65, 43-62.[Medline]
Oda, H., Uemura, T., Harada, Y., Iwai, Y. and Takeichi, M. (1994). A Drosophila homolog of cadherin associated with Armadillo and essential for embryonic cell-cell adhesion. Dev. Biol. 165, 716-726.[Medline]
Ohsugi, M., Butz, S. and Kemler, R. (1999). Beta-Catenin is a major tyrosine-phosphorylated protein during mouse oocyte maturation and preimplantation development. Dev. Dynam. 216, 168-176.[Medline]
Orsulic, S. and Peifer, M. (1996). An in vivo structure-function study of armadillo, the beta-Catenin homologue, reveals both separate and overlapping regions of the protein required for cell adhesion and for wingless signaling. J. Cell Biol. 134, 1283-1300.[Abstract]
Ozawa, M. and Kemler, R. (1998). Altered cell adhesion activity by pervanadate due to the dissociation of alpha-Catenin from the E-cadherin-catenin complex. J. Biol. Chem. 273, 6166-6170.
Patel, N. H., Snow, P. M. and Goodman, C. S. (1987). Characterization and cloning of fasciclin III: a glycoprotein expressed on a subset of neurons and axon pathways in Drosophila. Cell 48, 975-988.[Medline]
Peifer, M. (1993). The product of the Drosophila segment polarity gene armadillo is part of a multi-protein complex resembling the vertebrate adherens junction. J. Cell Sci. 105, 993-1000.
Peifer, M., Pai, L. M. and Casey, M. (1994). Phosphorylation of the Drosophila adherens junction protein Armadillo: roles for wingless signal and zeste-white 3 kinase. Dev. Biol. 166, 543-556.[Medline]
Peifer, M. and Polakis, P. (2000). Wnt signaling in oncogenesis and embryogenesis a look outside the nucleus. Science 287, 1606-1609.
Provost, E. and Rimm, D. L. (1999). Controversies at the cytoplasmic face of the cadherin-based adhesion complex. Curr. Opin. Cell Biol. 11, 567-572.[Medline]
Queenan, A. M., Ghabrial, A. and Schüpbach, T. (1997). Ectopic activation of torpedo/Egfr, a Drosophila receptor tyrosine kinase, dorsalizes both the eggshell and the embryo. Development 124, 3871-3880.
Redies, C. (2000). Cadherins in the central nervous system. Prog. Neurobiol. 61, 611-648.[Medline]
Sanson, B., White, P. and Vincent, J. P. (1996). Uncoupling cadherin-based adhesion from wingless signalling in Drosophila. Nature 383, 627-630.[Medline]
Steller, H., Fischbach, K. F. and Rubin, G. M. (1987). Disconnected: a locus required for neuronal pathway formation in the visual system of Drosophila. Cell 50, 1139-1153.[Medline]
Sullivan, W. and Theurkauf, W. E. (1995). The cytoskeleton and morphogenesis of the early Drosophila embryo. Curr. Opin. Cell Biol. 7, 18-22.[Medline]
Takahashi, K., Suzuki, K. and Tsukatani, Y. (1997). Induction of tyrosine phosphorylation and association of beta-Catenin with EGF receptor upon tryptic digestion of quiescent cells at confluence. Oncogene 15, 71-78.[Medline]
Tautz, D. and Pfeifle, C. (1989). A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98, 81-85.[Medline]
Tepass, U., Theres, C. and Knust, E. (1990). crumbs encodes an EGF-like protein expressed on apical membranes of Drosophila epithelial cells and required for organization of epithelia. Cell 61, 787-799.[Medline]
Tepass, U. and Hartenstein, V. (1994). The development of cellular junctions in the Drosophila embryo. Dev. Biol. 161, 563-596.[Medline]
Tepass, U., Gruszynski-DeFeo, E., Haag, T. A., Omatyar, L., Torok, T. and Hartenstein, V. (1996). shotgun encodes Drosophila E-cadherin and is preferentially required during cell rearrangement in the neurectoderm and other morphogenetically active epithelia. Genes Dev. 10, 672-685.[Abstract]
Tepass, U. (1999). Genetic analysis of cadherin function in animal morphogenesis. Curr. Opin. Cell Biol. 11, 540-548.[Medline]
Tepass, U., Truong, K., Godt, D., Ikura, M. and Peifer, M. (2000). Cadherins in embryonic and neural morphogenesis. Nat. Rev. Mol. Cell. Biol. 1, 91-100.[Medline]
Tepass, U., Tanentzapf, G., Ward, R. and Fehon, R. (2001). Epithelial cell polarity and cell junctions in Drosophila. Annu. Rev. Genet. 35, 747-784.[Medline]
Tsukatani, Y., Suzuki, K. and Takahashi, K. (1997). Loss of density-dependent growth inhibition and dissociation of alpha-Catenin from E-cadherin. J. Cell. Physiol 173, 54-63.[Medline]
Uemura, T., Oda, H., Kraut, R., Hayashi, S., Kotaoka, Y. and Takeichi, M. (1996). Zygotic Drosophila E-cadherin expression is required for processes of dynamic epithelial cell rearrangement in the Drosophila embryo. Genes Dev. 10, 659-671.[Abstract]
Ungar, A. R., Kelly, G. M. and Moon, R. T. (1995). Wnt4 affects morphogenesis when misexpressed in the zebrafish embryo. Mech. Dev. 52, 153-164.[Medline]
White, K., Grether, M. E., Abrams, J. M., Young, L., Farrell, K. and Steller, H. (1994). Genetic control of programmed cell death in Drosophila. Science 264, 677-683.[Medline]
Woods, D. F., Wu, J. W. and Bryant, P. J. (1997). Localization of proteins to the apico-lateral junctions of Drosophila epithelia. Dev. Genet. 20, 111-118.[Medline]
Yagi, T. and Takeichi, M. (2000). Cadherin superfamily genes: functions, genomic organization, and neurologic diversity. Genes Dev. 14, 1169-1180.
Younossi-Hartenstein, A., Green, P., Liaw, G., Rudolph, K., Lengyel, J. and Hartenstein, V. (1997). Control of early neurogenesis of the Drosophila brain by the head gap genes tll, otd, ems, and btd. Dev. Biol. 182, 270-283.[Medline]
Zak, N. B., Wides, R. J., Schejter, E. D., Raz, E. and Shilo, B. Z. (1990). Localization of the DER/flb protein in embryos: implications on the faint little ball lethal phenotype. Development 109, 865-874.[Abstract]
Zipursky, S. L., Venkatesh, T. R., Teplow, D. B. and Benzer, S. (1984). Neuronal development in the Drosophila retina: monoclonal antibodies as molecular probes. Cell 36, 15-26.[Medline]