canoe
A Ras-interacting protein with molecular mass of about 180 kDa (p180) was partially purified from bovine brain membrane extract by Ha-Ras (Drosophila homolog Ras85D) affinity column chromatography. This protein binds to a GTP Ha-Ras affinity column, but not to a column
containing GDP-Ha-Ras. or to a column made with Ras that contains a mutation in the effector domain (Ha-RasA38). The amino acid sequences of the peptides derived from p180 are almost identical to
those of human AF-6; AF-6 has been identified as the fusion partner of the ALL-1 protein. The
ALL-1/AF-6 chimeric protein is the critical product of the t (6:11) abnormality associated with some human leukemia. AF-6 has a GLGF/Dlg homology repeat (DHR) motif and shows a high degree of sequence similarity to Drosophila Canoe, which is assumed to function downstream from Notch in a common developmental
pathway. The recombinant N-terminal domain of AF-6 and Canoe specifically
interact with GTP Ha-Ras. The known Ras target c-Raf-1 inhibits
the interaction of AF-6 with GTP gamma Ha-Ras. These results indicate that
AF-6 and Canoe are putative targets for Ras (Kuriyama, 1996). The direct interaction of Raf, phosphatidylinositol-3-OH kinase, Ral GDS and Rin1 with activated Ras has been demonstrated. There is no obvious homology with AF-6 or Canoe among the Ras-interacting interfaces of these proteins, indicating that activated Ras can recognize a variety of target interfaces (Kuriyama, 1996 and references).
The interaction between AF-6 and Ras was also noted by Van Aelst (1994).
canoe and polychaetoid genetically interact giving rise to a more severe dorsal closure phenotype than one resulting from canoe mutation alone. canoe3 is an embryonic lethal allele of cno and displays a typical dorsal open phenotype. cnomis1 is a weak hypomorph that yields adult flies with rough eyes and subtle changes in the bristle number. In a search for mutations that interact with cnomis1, polychaetoid was identified as an enhancer of cno phenotypes. Flies doubly homozygous for pydtam1 and cnomis1 die as embryos; this represents a synthetic lethal combination. Examination of embryonic cuticles demonstrates that the cnomis1;pydtam1 double mutant remains open dorsally. Comparisons of cell shape during dorsal closure reveal that cno3 embryos exhibit insufficient elongation of cells. This is most evident in the leading edge cells, which appear square in cno3, in contrast to the oblong cells of wild-type. cnomis1;pydtam1 double mutant embryos exhibit a more extreme phenotype than single mutants: the leading edge cells elongate even less than in cno3 mutants. These results suggest that cno and pyd are required for coordinated cell shape changes in the cells of the leading edge and the lateral ectodermal cells during dorsal closure (Takahashi, 1998).
There is compelling evidence that the small GTPase Drac1 functions in dorsal closure as an upstream (early acting) element of the JNK pathway, which is composed of hemipterous, basket and puckered. To determine if cno is further upstream of Drac1, puckered-lacZ expression was examined in cno3 homozygous embryos: the leading edge of the epidermis in these embryos is driven to express Drac1V12, a constitutively active form of Drac1. If Drac1 is upstream of cno, then the effect of Drac1V12 on puc-lacZ transcription should be blocked by the loss of cno function. Targeted expression of Drac1V12 in the leading edge cells restores puc-lacZ transcription in cno3 homozygotes to a level comparable to that of wild-type. This result is compatible with the hypothesis that cno is upstream of Drac1, or that cno functions in a pathway parallel to that of Drac1 (Takahashi, 1998).
Demonstration of a physical interaction between Cno and Pyd places Pyd similarly upstream of Rac in the dorsal closure pathway. Cno and Pyd exhibit a similar tissue distribution and appear to colocalize at junctional membrane sites within the cell. ZO-1 is a component of both tight junctions and adherens junctions in mammalian cells. Mammalian ZO-1 binds to alpha-spectrin, which cross-links with actin filaments, thereby affecting cell shape. Pyd and mammalin ZO-1 also interact with Drosophila Cortactin and mammalian cortactin respectively.
Mammalian Cortactin is known to be a filamentous actin cross-linking protein and a substrate of Src protein tyrosine kinase. Cortactin is phosphorylated at tyrosine
residues upon stimulation by extracellular signals. Filamentous actin cross-linking activity
of cortactin is attenuated by Src. The intracellular localization of mammalian cortactin is regulated by the activation
of Rac1. Cortactin redistributes from the cytoplasm into membrane ruffles as a result of growth factor-induced Rac1 activation, and
this translocation is blocked by expression of dominant negative Rac1N17. Thus in mammals, cortactin is a putative target of Rac1-induced
signal transduction events involved in membrane ruffling and lamellipodia formation. It would thus seem that Rac signaling is tied to actin dynamics and Polychaetoid/ZO-1 function both in Drosophila and mammals (Takahashi, 1998 and references).
The physical and genetic interactions of Pyd and Canoe proteins, and the genes that code for them, respectively, are interesting in light of the genetic interaction between canoe and Notch. Cno has a DHR motif, a conserved sequence associated with protein interaction found in Discs large and Polychaetoid. The molecular structure of Cno suggests its direct association with Ras. Cno has significant homology with a mammalian Ras-binding protein AF-6 (Kuriyama, 1996). cno interacts genetically with the split allele of Notch, for eye, bristle and wing development. An interrupted wing vein in Ax1, one N allele producing an activated form of Notch protein, is dominantly suppressed by cno mutation (Miyamoto, 1995). What exactly is the biochemical pathway leading to extra bristles in polychaetoid and canoe mutation and how might this pathway intersect with the Notch pathway? What is the connection between Pyd, Cno and Rac leading to the activation of the JNK pathway during dorsal closure? These questions await future experimentation (Takahashi, 1998).
Rap1 belongs to the highly conserved Ras subfamily of small GTPases. In Drosophila, Rap1 plays a critical role in many different morphogenetic processes, but the molecular mechanisms executing its function are unknown. Canoe (Cno), the Drosophila homolog of mammalian junctional protein AF-6, has been shown to act as an effector of Rap1 in vivo. Cno binds to the activated form of Rap1 in a yeast two-hybrid assay, the two molecules colocalize to the adherens junction, and they display very similar phenotypes in embryonic dorsal closure (DC), a process that relies on the elongation and migration of epithelial cell sheets. Genetic interaction experiments show that Rap1 and Cno act in the same molecular pathway during DC and that the function of both molecules in DC depends on their ability to interact. Rap1 acts upstream of Cno, but Rap1, unlike Cno, is not involved in the stimulation of JNK pathway activity, indicating that Cno has both a Rap1-dependent and a Rap1-independent function in the DC process (Boettner, 2003).
Rap1 cycles between an inactive GDP-bound and an active GTP-bound state, eliciting distinct downstream responses in the active state. Mammalian Rap proteins were originally identified as antagonists of oncogenic Ras, but more recent studies suggest that the function of Rap1 is largely independent of Ras. While Ras is mainly localized at the plasma membrane, Rap1 has been found in different membrane compartments, depending on the cell type. Further, Rap1 activation appears to be stimulated by numerous exchange factors that do not act on the prototypic Ras GTPases. Rap1 has been shown to act in a Ras-independent manner in the production of superoxide, in cAMP-induced neurite outgrowth, and, most recently, in the regulation of integrin-mediated cell adhesion and AMPA receptor trafficking during synaptic plasticity (Boettner, 2003 and references therein).
Perhaps the most important insights into the function of Rap1 are emerging from studies in Drosophila. Loss-of-function (lof) mutations in Drosophila Rap1 cause severe morphogenetic abnormalities during embryonic development, while cell proliferation and cell fate determination, processes that rely heavily on regulation by Ras, appear to be unaffected. Specifically, the ventral invagination and migration of mesodermal precursors in the embryo are severely impaired, as are head involution, dorsal closure, and the migration of gonadal precursors (Asha, 1999). More recently, Rap1 has been shown to play a role in cell adhesion, specifically in the positioning of adherens junctions in proliferating epithelial cells (Knox, 2002). These findings strongly suggest that Rap1 plays a largely Ras-independent role in cell migration and morphogenesis (Boettner, 2003 and references therein).
Little is currently known about the signaling pathways mediating the downstream effects of Rap1 in vertebrates or Drosophila. A number of molecules that were originally identified in vertebrates as Ras-interacting proteins, including B-Raf, members of the RalGEF family, and AF-6, were subsequently shown to associate with Rap1 as well. However, the relevance of these interactions for Rap1 function in vivo remains largely unknown; to date, none of these molecules have been shown to act as Rap1 targets in an in vivo context (Boettner, 2003 and references therein).
This study reports that Canoe (Cno), the Drosophila ortholog of AF-6, acts as an effector of Rap1 during dorsal closure (DC) of the Drosophila embryo. DC is a morphogenetic process that occurs during midembryogenesis and involves the dorsalward movement of the lateral ectoderm over the amnioserosa, a transient structure that covers the dorsal aspect of the embryo, to enclose the embryo. This process relies entirely on the migration and elongation of ectodermal cells, without cell recruitment or proliferation, and is akin to the epithelial cell sheet movements that occur during wound healing. Among the genes identified as necessary for normal DC are proteins associated with the cytoskeleton and/or cell junctions and components of the Drosophila Jun N-terminal kinase (JNK) and Decapentaplegic (Dpp) pathways. cno is required for DC; its protein is localized to the adherens junction and feeds into the JNK pathway by an unknown mechanism. Apart from the fact that it interacts with the ZO-1 homolog Tamou, nothing is known about the regulation of Cno activity at the adherens junction (Boettner, 2003).
Cno has been identified as a protein that interacts with activated Rap1 in a yeast two-hybrid screen. To address the physiological relevance of this interaction, localization studies, a comparative phenotypic analysis, and genetic interaction experiments were undertaken for the two proteins. Rap1 and cno loci are shown to interact synergistically in DC and the physical interaction between Rap1 and Cno is required for DC. The role of Canoe in promoting JNK pathway activity is independent of Rap1 and Canoe therefore has two separate functions in DC (Boettner, 2003).
In Drosophila, embryos lacking both zygotic and maternal Rap1 display strong defects in diverse morphological aspects of embryogenesis, such as ventral invagination, migration of mesodermal precursors, head involution, and DC. A key question is which effector pathways mediate the morphogenetic functions of Rap1. The yeast two-hybrid system was used to identify Drosophila Rap1-specific effector molecules from an embryonic library and several cDNAs encoding Cno were retrieved. Both N-terminal Ras-binding domains (RA1 and RA2) of Cno possess Rap1-binding potential and they interact only with a constitutively active Rap1 mutant, Rap1V12, but not with a dominant negative version of Rap1, Rap1N17, suggesting that Cno may act as an effector for Rap1 (Boettner, 2003).
Several lines of evidence are provided confirming this hypothesis. Rap1 and Cno partially colocalize at the adherens junction in the two tissues that are involved in DC, the amnioserosa and the lateral ectoderm, with Rap1 being present at the entire lateral membrane and also showing vesicular expression throughout the cytoplasm. Moreover, loss of function of the two molecules leads to similar phenotypes, at both the cuticular and the cellular level. To directly address the question whether Rap1 utilizes Cno as an effector during DC, a series of genetic experiments were conducted. They demonstrate that the two molecules act in the same pathway and their physical interaction is essential for their function in DC: (1) Removal of zygotic Rap1 strongly enhances the phenotype of a weak heteroallelic cno combination; (2) removal of the RA-interaction domains and, thus, removal of the ability to bind Rap1, reduces the ability of cno transgenes to rescue the cno lof phenotype, and (3) removal of the RA-interaction domains eliminates the ability of cno to rescue Rap1N17. Finally, the finding that activated Rap1V12 fails to rescue the cno lof defects indicates that Rap1 acts upstream of Cno. Taken together, the yeast two-hybrid data, colocalization results, and genetic interaction experiments provide comprehensive evidence that Cno functions as a downstream effector of Rap1 in the DC process. These findings represent the first demonstration of a protein acting as a Rap1 effector in vivo (Boettner, 2003).
The events downstream of Rap1 and Cno, however, appear to be more complex. Several independent findings suggest that Cno's role in DC can be separated into Rap1-independent and Rap1-dependent functions: removal of the RA-interaction domains does not affect the ability of the remainder of the protein to localize to the adherens junction, and the mutant protein retains the capacity to partially rescue the DC defect of a cno lof mutant. Further, Cno feeds into the JNK pathway, while Rap1 does not: dpp expression levels in the LE are significantly reduced in cno lof embryos at later stages of DC, but appear unaffected in Rap1 mutants. In addition, cno lof is partially rescued by overexpressing bsk (DJNK), whereas the Rap1N17 defect is not. Given the multidomain structure of Cno, it is not surprising that the molecule would participate in multiple pathways. Such a bifurcation of the pathway would also explain the lack of transitivity observed in rescue experiments: Rap1 lof is (partially) rescued by cno overexpression, cno lof is (partially) rescued by bsk overexpression, but Rap1 lof is not rescued by bsk overexpression. The fact that both cnoDeltaN and bsk are unable to rescue Rap1 lof demonstrates that the Rap1-independent function of Cno cannot compensate for the loss of Rap1. This leaves the reciprocal question of whether Rap1 may have a second, Cno-independent function in DC. The fact that the DC phenotype of Rap1N17 is as severe as that of cno lof without affecting JNK pathway signaling might suggest that Rap1 has additional effectors in DC (as does the fact that the phenotype of Rap1N17 is more severe than that of cno2; ptcGAL4 UAScnoDeltaN). However, no conclusive evidence has been found to support this idea, since the additional effectors of Rap1 identified in the yeast two-hybrid screen have not been investigated for their role in DC (Boettner, 2003).
One obstacle in investigating the function of Rap1 is its pleiotropy. A detailed analysis of DC defects, in particular, is difficult to perform in Rap1 null embryos, due to the severe disruption of multiple aspects of embryonic development prior to DC. Therefore, use was made of the dominant negative Rap1N17 mutant. When expressed at appropriate stages in the epithelial cells that are involved in the DC process, this transgene results in robust DC defects. However, early in vitro studies appeared to show that the Rap1N17 mutant does not compete well with normal Rap1 for the GEF C3G, calling into question whether this mutant protein can be regarded as a Rap1 dominant negative. But in vivo studies using mammalian Rap1 and now the current study clearly show that Rap1N17 acts as a dominant negative mutant in Rap1 signaling. The successful rescue of Rap1N17 with a concomitantly expressed Rap1wt transgene demonstrates the specificity of the mutant. Further, dominant negative versions of Drosophila Ras1 and Ras2, the counterparts of the mammalian H, K, and N-Ras and of the R-Ras proteins, respectively, do not disrupt DC when they are examined under the same conditions. This shows that the interaction between DRas1 and Cno detected in vitro and the genetic interaction between DRas1 and Cno that influences cone cell formation in the Drosophila eye, have no role during DC (Boettner, 2003).
On which cellular processes might Rap1 and Cno act? Cno is a multidomain protein consisting of several known and putative protein-interaction domains, including the two RA domains and a PDZ domain, which targets proteins to specific cell membranes and assembles proteins into supramolecular signaling complexes, but no catalytic domain. Cno localizes to the adherens junction and may act by localizing and clustering signal transduction components at the junction or by modulating the mechanical resistance of the adherens junction, and thus, directly or indirectly, influence JNK signaling. Since Cno is found at the adherens junctions under Rap1 lof conditions as well as in the absence of its RA domains, Rap1 cannot be required for the initial localization of the Cno protein, suggesting that Rap1 influences the activity of Cno by changing its conformation. However, another possibility is suggested by a study by Knox (2002), where it was found that Rap1 function is required for evenly (re-)distributing adherens junction components in wing disc epithelial cells after mitosis. It is likely that the adherens junctions in the cells that undergo stretching in the embryonic ectoderm during DC are similarly subject to dynamic reorganization, which may in part be regulated by the Rap1/Cno complex. This idea would be consistent with the observation that in Rap1 and cno lof mutants the lateral ectoderm begins its dorsal stretching, but is then unable to complete the process. Interestingly, Rap1 in mammalian cells has been shown to be activated in cell-stretching assays. In this system, force initiation apparently results in the activation of the JNK kinase family member p38, suggesting the existence of a Rap1-dependent 'mechanosensory' pathway. The data fit this idea. Future studies using fluorescently tagged Rap1 and Cno proteins and live imaging will shed light on dynamic aspects of their localization and function during DC (Boettner, 2003).
Echinoid is an immunoglobulin domain-containing transmembrane protein that
modulates cell-cell signaling by Notch and the EGF receptors. In
the Drosophila wing disc epithelium, Echinoid is a component of adherens
junctions that cooperates with DE-Cadherin in cell adhesion. Echinoid and
β-catenin (a DE-Cadherin interacting protein) each possess a C-terminal
PDZ domain binding motif that binds to Bazooka/PAR-3; these motifs redundantly
position Bazooka to adherens junctions. Echinoid also links to actin filaments
by binding to Canoe/AF-6/afadin. Moreover, interfaces between
Echinoid- and Echinoid+ cells, like those between
DE-Cadherin- and DE-Cadherin+ cells, are deficient
in adherens junctions and form actin cables. These characteristics probably
facilitate the strong sorting behavior of cells that lack either of these
cell-adhesion molecules. Finally, cells lacking either Echinoid or DE-Cadherin
accumulate a high density of the reciprocal protein, further suggesting that
Echinoid and DE-Cadherin play similar and complementary roles in cell adhesion (Wei, 2005).
Several observations prompted the study of Ed as a canonical CAM in the
monolayered wing imaginal disc. Thus, mitotic recombination clones of cells
mutant for the null allele ed1x5 exhibit rounded and smooth
contours, in contrast to clones of wild-type
cells that show wiggly shapes. This indicated that
ed- /- cells have distinct
adhesive properties and assort with themselves rather than with the surrounding
ed+/- M+/- cells.
(ed1x5 clones were M+,
since without a growth advantage they hardly survive). It was also observed that Ed was absent
from the membrane of the heterozygous cells that contacted the mutant cells,
a finding consistent with the
observation that Ed forms homophilic interactions and that these are required to
incorporate/stabilize Ed at the cell membrane. Finally, Ed was found to
localize basally to the apical marker Crb and apically to the basolateral marker Dlg.
In fact, Ed colocalizes with both DE-Cad and Arm,
and, therefore, it might be part of AJs. AJs are structures important for
cell-cell contact and recognition. So, these results suggested that Ed plays a
role in cell-cell adhesion (Wei, 2005).
Whether Ed affects components of AJs was examined by analyzing the localization of Arm within ed mutant clones. Arm strongly accumulates at the apical membranes of ed- /- cells, and these cells
have a reduced apical surface.
Both effects are clear in small clones, but cells within larger clones (over hundreds of cells) had both
the density of Arm and the apical surface more similar to those of the wild-type
cells. Similar observations were made with DE-Cad and Actin. It is suggested that the increased concentration of these molecules in small clones most probably results from the apical constriction as supported by the accumulation of nonmuscle myosin II, without a net per cell increment of these proteins.
Alternatively, it could result from increased stability of these proteins. The
apical constriction continued through the SJs and ended at the planes just below
the GJs as revealed by an Innexin antibody. Hence, these
ed- /- cells adopt a bottle
shape. In contrast, the apposed ed- /- and
ed+/- cells that form the border of the clone enlarge
and adopte a rectangular shape. At this interface, the
ed- /- cells often contacted the
heterozygous cells by their long sides, as if in an attempt to minimize the number
of cells that formed the interface (Wei, 2005).
Interestingly, Arm and DE-Cad, but not Actin,
are depleted at the interface membrane of both small and large clones. This suggests that ed- /- and ed
heterozygous cells discriminate one another and that AJs do not form properly in
between them (Wei, 2005).
ed clones are surrounded by an Actin 'cable'. High-magnification images
suggest that the cable is contained within the ed heterozygous cells
surrounding the clone and that it is therefore generated by these cells. Several observations
suggest that this Actin cable exerts a force. The cells surrounding an ed
clone elongate toward the clone and accumulate nonmuscle myosin II at the
interface membrane, as if attempting to cover the space exposed by the apically constricted
ed- /- cells. This effect is
reminiscent of the stretching of the leading-edge cells that will cover the
underlying amnioserosa during dorsal closure of the embryos. In the wing disc,
the boundary that separates the dorsal (D) and ventral (V) regions of the wing
pouch has the shape of a smooth arc and contains an actin 'fence'.
After the second instar, this boundary corresponds to a compartment border that
imposes absolute restrictions to cell lineages. Large
ed- /- clones close to or
touching this boundary displace it toward the clones. In contrast, ed clones that
straddle the boundary do not overtly distorted it, although the boundary could be less
smooth within the clone. (Straddling clones
might be originated before the compartment border was established or might be
formed of D and V clones that fuse together). Moreover, the Actin
cable surrounding the clones fuse with the Actin fence at the D/V
boundary, suggesting that the distortion of this boundary is effected through
this Actin linkage. Control
ed+ M+
clones do not induce such distortions. These observations
suggest that the Actin cable may contribute to the roundish shape of the
ed clones and help confine their cells (Wei, 2005).
DE-Cad is a classical homophilic cell adhesion molecule of
AJs. It interacts with β-catenin/Arm, which in turn binds α-catenin.
Through the association between α-catenin and F-Actin, DE-Cad establishes
links between cells that connect to the Actin cytoskeleton. This study shows that Ed
is another CAM that, at the resolution of confocal microscopy, is
also located at the AJs of imaginal disc cells. While cells in clones mutant for
ed still seem to form normal AJs, the cells at the border of the clone
seem impaired in forming them. It is hypothesized that this may help them segregate
from surrounding ed+/- cells. Ed was identified as a
binding partner for PDZ proteins that, similarly to Arm, helps localize Baz to
AJs. Moreover, it was found that through the binding of Cno, Ed, like
DE-Cad/β-catenin, may link to F-Actin. Hence, Ed has functions in
cell-cell adhesion similar to those of DE-Cad (Wei, 2005).
The differential adhesion hypothesis proposes that cell sorting may be driven by differences in
the quantity and/or quality of adhesive molecules displayed on the surface of
cells. In keeping with this hypothesis, it was found that
ed- /- cells sort out from
ed+/- cells, as shown by the remarkably round shapes
and smooth contours of the ed clones. Moreover, their differential
adhesiveness is also manifest by the fusion of different ed clones to
yield composite but still roundish clones. It is suggested that contraction of the
apically enriched Actin network and of the actin cable surrounding the clone,
possibly by interaction with nonmuscle myosin II also present there, may
contribute to the the apical constriction of the
ed- /- cells. It was also observed
that the interface between ed+/- and
ed- /- cells is depleted of
DE-Cad, Arm and Baz, besides completely lacking Ed. This strongly suggests that
this interface is deficient in AJs and probably helps to insulate
ed- /- cells from the
surrounding ed heterozygous cells. It is hypothesized that this deficiency of
AJs, which may reduce adhesion between ed+/- and
ed- /- cells, and
the inward-pulling force generated by apical constriction and the actin cable
may help create the smooth and rounded contour of the clones at the level of
AJs. At the plane of SJs, the clonal boundary is not as smooth. This may be due
to the presence of normal levels of SJs, since seemingly wild-type amounts of
Dlg were detected at the interface membrane. Normal levels of SJs may allow the
clones to remain integrated in the epithelium. It is stressed that when ed
clones grow large, the apical constriction disappears, suggesting that the
forces responsible for this constriction become insufficient or no longer
operate. If the force is exerted, at least in part, by the Actin cable
surrounding the clone, as in a purse-string mechanism, it would make sense that
this force becomes ineffectual as the number of cells within the clone
increases. Remarkably, these differences of apical cell constriction observed in
small and large ed clones have a correlate on the adult wing blade: small
clones display an increased density of trichomes, implying that their cells are
small or more tightly packed, whereas large clones have cells of normal size.
This indicates that the apical constriction is retained through imaginal disc
eversion, when the disc epithelium changes from columnar to planar (Wei, 2005).
In the embryonic
epithelium, Baz, localized to both AJs and the marginal zone, is the initial
apical regulator. How
is Baz recruited to the apical domain? In the follicular epithelium, Baz is
localized to this domain through lateral exclusion mediated by PAR-1/14-3-3 and
apical anchoring by Crb/Sdt/Patj. The data support an additional mechanism to localize
Baz to the apical domain. Both Ed and Arm can bind Baz through their C-terminal
PDZ binding motif and therefore they may redundantly localize Baz to AJs.
Indeed, the localization of Baz to AJs is relatively normal in the absence of
either one. Most Baz is lost only when both Arm and Ed are depleted, as
occurs at the interface membrane of ed clones or in large shg
clones where Ed gradually breaks down. In the latter case, there is good
colocalization between Baz and the sites maintaining residual Ed. It is suggested
that in the epithelium of the wing disc, Baz localizes to AJs by the combined
effects of its binding to Ed/Arm and the lateral exclusion of PAR-1/14-3-3.
Additionally, apical anchoring of Baz may be mediated by direct association
between the Baz and Crb apical complexes. During early embyogenesis, Ed is also
present at pseudocleavage furrows. This observation,
together with the ability of Ed to localize Baz to AJs, may explain the finding
that during cellularization, Baz can accumulate apically in the absence of Arm.
Ed also binds to the PDZ domain of Cno and mediates its localization to AJs, where Cno
interacts with F-Actin either directly or indirectly through the association
with Polychaetoid/ZO-1. Interestingly, the evolutionally conserved EIIV domain
of Ed binds Baz and Cno in a mutually exclusive manner. Thus, the concentrations
of and differential affinities between Ed, Baz, and Cno should determine their
dynamic equilibrium at AJs (Wei, 2005).
Although Baz is critical to form AJs in the
blastoderm and in the follicular epithelium, removal of Baz
(or Par-6) from cells of the wing disc does not affect the
localization of DE-Cad or Ed to AJs. This is consistent with the report that in
imaginal discs, Baz does not affect the localization of DE-Cad and Dlg but is
required for the asymmetric localization of cell fate determinants. Together, these
results suggest that in wing discs, the Baz complex is not critical for the
formation of AJs, and that the effect of the loss of Ed on AJs
formation/maintenance is not due to Baz depletion (Wei, 2005).
Several similarities between the roles of DE-Cad and Ed in the
wing disc epithelium are worth noting. Both Ed and DE-Cad are CAMs that
establish homophilic interactions and localize to AJs. The absence of either Ed
or of DE-Cad in cells of small clones causes their apical constriction and
strong segregation from wild-type cells, giving rise to smooth round borders. In
both cases, the mutant cells are impaired in forming AJs with neighboring
wild-type or heterozygous cells and are surrounded by an Actin cable. Ed
interacts with Cno, and DE-Cad with Arm, and both Cno and Arm directly or
indirectly associate with F-Actin. Thus, Ed and DE-Cad represent two distinct
classes of CAMs, with widely different chemical compositions, that connect to
F-Actin, contribute to cell adhesion in the wing disc, and seem to have
partially overlapping functions (Wei, 2005).
In contrast, DE-Cad and Ed differ in
their ability to regulate the apical/basal cell polarity. Ed affects components
of AJs, but not those of the apical Crb and the basolateral Dlg complexes. In
contrast, DE-Cadherin is necessary for Crb localization, but similarly to Ed, it
is not required for Dlg localization. Furthermore, the maintenance of Ed at AJs
requires DE-Cad. In contrast, localization of DE-Cad to AJs is independent of
Ed. Interestingly, the DE-Cad/Arm complex is not essential for the formation of
the follicular epithelium, but upon removal of this complex, the integrity of the
epithelium is lost slowly over the period of several days. This suggests that
other molecules may be maintaining the epithelial structure. During stages
1 to 10 of oogenesis Ed is mainly expressed in the follicle cells, and these cells,
if mutant for ed, show at low frequency a multilayered structure with
disrupted expression of some polarity markers. Thus,
it will be of interest to elucidate whether, in this epithelium, Ed and
DE-Cad/Arm also play partially redundant roles in cell adhesion and apical/basal
polarity. While both Ed and DE-Cad contribute to cell adhesion and recognition,
it is unclear whether each molecule imparts specific recognition properties to
cells, so that the final cell-cell affinity results from the sum of distinct
affinities mediated by these different CAMs. More specifically, can an increased
level (density) of DE-Cad replace the absence of Ed? The results showing that
ed- /- cells, with either normal
levels (in large clones) or high density (in small clones) of DE-Cad, do not
intermix with wild-type cells suggests that the binding specificity provided by a
given CAM is not overruled by a higher level (density) of a different CAM.
Moreover, the cell sorting properties conferred by Ed cannot account for the
separation of cells at both sides of the A/P compartment boundary of the wing
disc because A and P cells do not intermingle within composite ed, smo
double mutant clones. (Similarly, DE-Cad is not responsible for the sorting out
of A and P cells. Hence, cell-cell adhesion in the wing disc appears to depend on
multiple CAMs (Ed, DE-Cad, etc.), each imparting specific cell recognition
properties. Although Ed and its C-terminal EIIV motif are conserved in
invertebrates, no
clear vertebrate homolog with 7 Ig domains and a PDZ domain binding motif has
been found. Nectin1-4 comprises a family of 3 Ig domain-containing CAM that have
several differentially spliced forms and localize to AJs. Most spliced
forms share a conserved C-terminal E/A-X-Y-V that binds the PDZ domain of
Afadin. Moreover, this motif also interacts with Par-3, the vertebrate homolog
of Baz. In spite of these similarities, overexpression of either nectin 1-α or 3-α does not rescue the remarkable clonal phenotype of ed (Wei, 2005).
Cadherin-based adherens junctions (AJs) mediate cell adhesion and regulate cell shape change. The nectin-afadin complex also localizes to AJs and links to the cytoskeleton. Mammalian afadin has been suggested to be essential for adhesion and polarity establishment, but its mechanism of action is unclear. In contrast, Drosophila's afadin homologue Canoe (Cno) has suggested roles in signal transduction during morphogenesis. In this study Cno was completely removed from embryos, testing these hypotheses. Surprisingly, Cno is not essential for AJ assembly or for AJ maintenance in many tissues. However, morphogenesis is impaired from the start. Apical constriction of mesodermal cells initiates but is not completed. The actomyosin cytoskeleton disconnects from AJs, uncoupling actomyosin constriction and cell shape change. Cno has multiple direct interactions with AJ proteins, but is not a core part of the cadherin-catenin complex. Instead, Cno localizes to AJs by a Rap1- and actin-dependent mechanism. These data suggest that Cno regulates linkage between AJs and the actin cytoskeleton during morphogenesis (Sawyer, 2009).
In stage 5 embryos before cellularization, CNO mRNA distributes uniformly at a low level throughout the embryo, except for the pole cells. At the cellular blastoderm, signals are detected along the dorsal midline, where three intensely stained domains (anterior, central, and posterior) are descernible. In additon, three ectodermal stripes reminiscent of parasegmental expression of gap genes are seen clearly in the central domain of the embryo. In stage 7-10, expression is confined to the dorsal furrows and the posterior midgut rudiment. The pattern of mRNA distribution may imply that Cno is required for delamination of presumptive endodermal cells, a process that depends on control of adhesion between midgut epithelial cells. Ectodermal expression becomes evident at stage 10. At stage 13, focal stainings are detected near the attachment site of the midgut to the foregut and hindgut (Miyamoto, 1995).
Polychaetoid protein is localized at the cell-cell junction. Observation of the presumptive wing blade region of the wing imaginal disc reveals that Pyd has a more basal distribution compared with Shotgun. Pyd colocalizes with the scaffolding protein Canoe. In the cellular blastoderm (stage 5), the Cno protein is distributed diffusely in the cytoplasm, with significant accumulation at the apical surface. The cytoplasmic staining decreases before gastrulation. In stage 13 embryos undergoing germ-band retraction, marked accumulation of Cno is observed in the amnioserosa, with persistent expression of Cno in the lateral epidermis. The intense staining of the amnioserosa and the apposed edges of the lateral epidermis continues during dorsal closure. At this stage of embryogenesis, the trachea in each segment begins to elongate laterally to form the tracheal system across the segments. In addition, Cno is localized in Malpighian tubules, hindgut and the central nervous system. The tissue localization of Pyd is remarkably similar to that of Cno. It is present in the cytoplasm in the blastoderm stage embryo. In the later stages, Pyd is exclusively localized to cell boundaries. The epidermis, amnioserosa, the margin of the closing epidermis, the tracheal system, the Malpighian tubes, the hindgut and the CNS all express Pyd at high levels (Takahashi, 1998).
Ectodermal epithelium was examined for Pyd and Cno colocalization. The two proteins partially colocalize: Pyd expression is more widespread than Cno expression. The domain of Cno expression and that of Fas III expression are mutally exclusive, whereas the distributions of Arm and Drosophila alpha-catenin coincide with that of Cno. In contrast, Pyd is expressed in areas at which Fas III is localized. Fas III distribution is known to be restricted to septate junctions, and Drosophila alpha-catenin and Armadillo are confined to adherens junctions. Cno colocalizes with Arm but not with Fas III in the embryonic epidermis. Thus the results indicate that Pyd is present at both the septate and adherens junctions while Cno exists predominantly at adherens junctions (Takahashi, 1998).
CNO transcripts are expressed ubiquitously in eye-antennal discs, with higher levels of expression in the lateral edge region (Miyamoto, 1995).
Flies doubly mutant for cnomis1
and scabrous and those for cnomis1 and Notch always
have rumpled, downward curving wings. The Notch/cnomis1 double mutant flies also exhibit a "giant socket" phenotype. These phenotypes
are rarely observed in flies singly mutant for either cnomis1, scabrous
or Notch. The wing vein gaps caused by another Notch allele
producing an activated form of N protein, are dominantly suppressed by
cnomis1. Heterozygosity for shaggy and myospheroid
promotes formation of extra wing veins in cnomis1 homozygotes. The
genetic interactions suggest that cno participates with members
of the Notch pathway in regulating adhesive cell-cell interactions for
the determination of cell fate. Since there appears to be a direct physical
interaction between Notch receptor and Dishevelled,
providing a link between Notch and wingless signaling, perhaps Canoe plays
a role in modifying this interaction (Miyamoto, 1995). Cone cells are lens-secreting cells in ommatidia, the unit eyes that compose the
compound eye of Drosophila. Each ommatidium contains four cone cells derived from
precursor cells of the R7 equivalence group which expresses the gene sevenless (sev).
When a constitutively active form of Ras1 (Ras1V12) is expressed in the R7
equivalence group cells using the sev promoter (sev-Ras1V12), additional cone cells
are formed in the ommatidium. Expression of Ras1N17, a dominant negative form of
Ras1, results in the formation of 1-3 fewer cone cells than normal in the ommatidium.
The effects of Ras1 variants on cone cell formation are modulated by changing the
gene dosage at the canoe locus, which encodes a cytoplasmic protein with
Ras-binding activity. An increase or decrease in gene dosage potentiates the
sev-Ras1v12 action, leading to marked induction of cone cells. A decrease in cno+
activity also enhances the sev-Ras1N17 action, resulting in a further decrease in the
number of cone cells contained in the ommatidium. In the absence of expression of
sev-Ras1V12 or sev-Ras1N17, an overdose of wild-type cno (cno+) promotes cone
cell formation, while a significant reduction in cno+ activity results in the formation of
1-3 fewer cone cells than normal in the ommatidium. It is proposed that there are two
signaling pathways in cone cell development, one for its promotion and the other for its
repression; Cno is thought to function as a negative regulator for both pathways. It is also
postulated that Cno acts predominantly on a prevailing pathway in a given
developmental context, thereby resulting in either an increase or a decrease in the
number of cone cells per ommatidium. The extra cone cells resulting from the
interplay of Ras1v12 and Cno are generated from a pool of undifferentiated cells, normally fated to either develop into pigment cells or undergo apoptosis (Matsuo, 1997).
Egfr signaling is evolutionarily conserved and controls a variety of different cellular processes. In Drosophila these include proliferation, patterning, cell-fate determination, migration and survival. Evidence is provided for a new role of Egfr signaling in controlling ommatidial rotation during planar cell polarity (PCP) establishment in the Drosophila eye. Although the signaling pathways involved in PCP establishment and photoreceptor cell-type specification are beginning to be unraveled, very little is known about the associated 90° rotation process. One of the few rotation-specific mutations known is roulette (rlt) in which ommatidia rotate to a random degree, often more than 90°. rlt is shown to be a rotation-specific allele of the inhibitory Egfr ligand Argos; modulation of Egfr activity shows defects in ommatidial rotation. The data indicate that, beside the Raf/MAPK cascade, the Ras effector Canoe/AF6 acts downstream of Egfr/Ras and provides a link from Egfr to cytoskeletal elements in this developmentally regulated cell motility process. Evidence is provided for an involvement of cadherins and non-muscle myosin II as downstream components controlling rotation. In particular, the involvement of the cadherin Flamingo, a PCP gene, downstream of Egfr signaling provides the first link between PCP establishment and the Egfr pathway (Gaengel, 2003).
Since ommatidial rotation is a cell motility process requiring cytoskeletal
rearrangements, it was of interest to determine if effectors of Egfr other than the Raf/MAPK cascade play a role in this process. The Ras GTPase, the main transducer of Egfr signaling, can utilize distinct effectors in different contexts. In addition to nuclear signaling, mediated by the Raf/MAPK/Pnt cascade, Ras can affect cell growth and cytoskeletal rearrangements via its effectors Rgl/Ral, Phospho-inositol-3-Kinase (PI3K) and Canoe, whose human homolog (AF6) is known as the critical partner of ALL1 in a chimeric protein associated with
myeloid leukemia (Gaengel, 2003).
A direct canoe (cno) requirement in
ommatidial rotation was tested using LOF alleles. First, it was asked whether cno
heterozygosity interacts with the Star48-5/+
rotation phenotype. Strikingly, similar to the enhancement observed with
Egfr or Ras, the cno2/+ and
cno3/+ genotypes enhance the
S48-5/+ rotation phenotype.
cno is required for cone cell and photoreceptor differentiation and
thus clones of null and strong alleles cause a general disorganization of the eye and are difficult to analyze for rotation defects. However, the hypomorphic cnomis1 allele is subviable in trans to the strong alleles cno2 and cno3 with mildly rough eyes, allowing an analysis of ommatidial rotation. Eye sections of such transheterozygous cno flies (e.g. cnomis1/cno2) reveal severe rotation
defects. To test whether such defects are already observed at the time when rotation takes place, cno mutant third instar eye discs were analyzed. Strikingly,
rotation defects, comparable in strength to the stronger aos alleles,
are apparent in cno eye imaginal discs. The discs were
counterstained with anti-Elav to ensure that the photoreceptor
complement is normal in such cno mutant discs and the observed
rotation abnormalities are primary defects, which was indeed confirmed. A
similar analysis of Ral/Rgl is precluded by the lack of suitable alleles. In
summary, these data indicate that cno plays a critical role in
ommatidial rotation and acts as an effector of Egfr/Ras signaling in this
context (Gaengel, 2003).
Since ommatidial rotation is a cell biological event, it is probable that
among the main read-outs affected are cell-adhesion properties of the
precluster cells and effects on cytoskeletal elements. This is further
supported by observations that (1) Raf/MAPK-independent and thus
transcription-independent Egfr/Ras signaling pathways are important, and (2)
that canoe is required in this context. To address this further, two sets of experiments were performed. First, tests were performed for genetic interactions between the dosage-sensitive Star–/+ rotation phenotype and selected factors required in cell adhesion and cytoskeletal regulation; and second, whether cell-adhesion components
such as cadherins and integrins are normally localized in
aosrlt and cnoMis1 mutant
backgrounds was directly analyzed (Gaengel, 2003).
The Egfr/Ras/Cno link is intriguing for several reasons. The cno
gene was originally identified as a mutation affecting the dorsal closure
process during embryogenesis. Cno
shows a genetic and molecular link to Ras: it contains two Ras-interacting
domains and binds both WT Ras and activated Ras-V12. In
addition, Cno has been postulated to link cytoskeletal elements to cellular
junctions via its ability to bind actin, its
interaction with ZO-1/Pyd and its homology with kinesin and myosin-like
domains. Thus Cno could directly mediate an Egfr/Ras signal to
cytoskeletal and cell architecture elements through its association with
adherens junctions and its kinesin and myosin-like domains.
Interestingly, Zipper does not only show a similar interaction with
Star, like Cno, but it is also required during embryonic dorsal closure, and thus a more general Cno-Zipper link might exist in cell motility contexts (Gaengel, 2003).
A second interesting feature of cno is that it has been
genetically linked to sca and Notch signaling.
First, the phenotype of the sca1 allele is strongly
enhanced by cno–/+. Second, cno alleles
also display Notch-like phenotypes in the wing and a GOF
Notch allele, NotchAbruptex, is suppressed by
cno. Although the biochemical role of Sca remains obscure, it
has been linked to Notch, possibly as a Notch ligand, in several contexts. Thus, since sca has recently been implicated in ommatidial rotation, the link between Cno and Sca/Notch is intriguing. Taken together, Cno could serve as a factor integrating signaling input from different pathways, e.g., Egfr and Notch in this process, and relaying this to cytoskeletal elements. The Canoe link is also interesting from a disease point of view since its human homolog AF6 is the critical partner of ALL1 in a chimeric protein associated with myeloid leukemia. Thus, taken together, Cno could serve as a factor integrating signaling input from different pathways, e.g., Egfr and Notch in ommatidial rotation, and relaying this to the regulation of cell adhesion and cytoskeletal elements in the context of a developmental patterning process or disease (Gaengel, 2003).
Thus Egfr/Ras signaling plays a general role in the
regulation of ommatidial rotation. Canoe has been identified as an effector of Ras in this context. Although much is known about how ommatidial chirality and the associated R3/R4 cell-fate decision are regulated (Fz/PCP-Notch signaling), no clear link between the mechanistic aspects of ommatidial rotation and Fz/PCP signaling previously existed. This is the first link to be demonstrated between Egfr signaling and PCP genes, namely Fmi. A further connection between Egfr signaling and PCP establishment is provided by Zipper, which acts downstream of Fz/Dsh and Rok in wing PCP and modifies the Star rotation phenotype. The identification of the Egfr pathway and its regulation of Fmi/cadherin-mediated cell adhesion will serve as an important entry point to further such studies (Gaengel, 2003).
Directed cellular movements are a universal feature of morphogenesis in
multicellular organisms. Differential adhesion between the stationary and
motile cells promotes these cellular movements to effect spatial patterning of
cells. A prominent feature of Drosophila eye development is the
90° rotational movement of the multicellular ommatidial precursors within
a matrix of stationary cells. This study shows that the cell adhesion molecules
Echinoid (Ed) and Friend of Echinoid (Fred) act throughout ommatidial rotation
to modulate the degree of ommatidial precursor movement. It is proposed that
differential levels of Ed and Fred between stationary and rotating cells at
the initiation of rotation create a permissive environment for cell movement,
and that uniform levels in these two populations later contribute to stopping
the movement. Based on genetic data, it is proposed that ed and
fred impart a second, independent, `brake-like' contribution to this
process via Egfr signaling. Ed and Fred are localized in largely distinct and
dynamic patterns throughout rotation. However, ed and fred
are required in only a subset of cells -- photoreceptors R1, R7 and R6 -- for
normal rotation, cells that have only recently been linked to a role in planar
cell polarity (PCP). This work also provides the first demonstration of a
requirement for cone cells in the ommatidial rotation aspect of PCP (Fetting, 2009).
ed and fred also genetically interact with the PCP genes,
but affect only the degree-of-rotation aspect of the PCP phenotype.
Significantly, this study demonstrates that at least one PCP protein, Stbm, is required in R7 to control the degree of ommatidial rotation (Fetting, 2009).
This study demonstrates that ed and fred have partially
overlapping functions during the two phases of ommatidial rotation. It is proposed that different levels of Ed and Fred in rotating and non-rotating cells
modulate the adhesivity of these cells, a prerequisite for rotation to occur.
In the second phase, Ed and Fred are required in R1, R6, R7 and the cone
cells, where they are likely to regulate the Egf receptor to contribute to the
slowing of rotation (Fetting, 2009).
There are two phases of rotation distinguishable by the rate at which the
ommatidia rotate. The initial phase (rows 4-7) is fast, with ommatidia
rotating 10-15° per row, whereas rotation slows to 5-10° per row in
the slow phase (rows 7-15). The data demonstrate that Ed and Fred function
during both phases and that they play unique roles in each phase (Fetting, 2009).
In the first phase, it is proposed that the tight regulation of Ed and Fred
levels between rotating and stationary cells creates an environment that is
permissive to rotation. Immediately before rotation starts, Ed begins to be
endocytosed in the ommatidial precluster cells. Concurrently, Ed levels fall
dramatically in these cells while remaining high in the stationary interommatidial cells (IOCs), setting up an imbalance in Ed levels between these two populations of cells. It is proposed that the resulting differential adhesion between these two cell populations enables the rotating cells to slide past their stationary neighbors in accordance with Steinberg's differential adhesion
hypothesis (DAH) (Steinberg, 2007). The DAH suggests that cell populations maximize the strength of adhesive bonding between them and minimize the adhesive free energy, and use tension generated by adhesion between cells to drive events
such as cell rearrangements during morphogenesis. Cells with equivalent levels
of Ed (or Fred) adhere more tightly to one another and adhesion is reduced
between cells with different levels of Ed (or Fred), thereby enabling the two groups to slide past one another. In support of this hypothesis, artificially equalizing levels of Ed or Fred significantly slows rotation (Fetting, 2009).
The data are consistent with Ed and Fred playing two key
roles in the slow phase by both directly and indirectly (through Egfr
signaling) affecting the physical component of the process. It is suggested that in both cases the outputs produce adhesive forces that slow/stop rotation. Ed and
Fred are required in photoreceptors R1, R6 and R7 and the cone cells for
normal ommatidial rotation. These cells do not become fully integrated into
the ommatidial cluster until the second half of rotation. Furthermore, R1, R6
and R7 constitute the rotation interface until the cone cells are recruited,
at which point the cone cells co-opt this position and role. Consequently, Ed
and Fred are required in the right place (the subset of cells that lie at the
rotation interface) and at the right time (the slower phase of rotation) to
play a role in slowing rotation (Fetting, 2009).
It is proposed that Ed and Fred activity in R1, R6, R7 and the cone cells
regulates Egfr signaling in these cells to slow/stop rotation as follows. Egfr
signaling promotes rotation via the Ras/Cno and Ras/Mapk/Pnt effectors
(Brown, 2003; Gaengel, 2003), so its output must be dampened to slow rotation. Ed binds and inhibits the Egf receptor, whereas Fred binds Ed and interferes with this inhibition. Therefore, cooperation between Ed and Fred precisely titrates
Egfr activity in the cells in which Ed and Fred function. As R1, R6 and R7 are
recruited into the ommatidial cluster, Ed levels are high in these cells,
thereby decreasing Egfr signaling at their side of the rotation interface,
thus impeding rotation. This inhibitory role switches to the cone cells when
they are recruited, creating a new rotation interface (Fetting, 2009).
Rotation may be slowed through Egfr signaling activity via its effector
Cno, the fly homolog of Afadin/AF-6, an actin-binding adherens junction (AJ) protein. Afadin and its binding partners, nectins and α-actinin, build and stabilize dynamic AJs that undergo remodeling (Ooshio, 2007). The majority of cno mutant ommatidia over-rotate, indicating that Cno inhibits ommatidial rotation. Since Egfr signaling promotes and Cno inhibits rotation, Egfr signaling is likely to suppress Cno activity during rotation thereby blocking stable junction formation. In this scenario, high levels of Egfr
would be required during the early phase of rotation to prevent Cno from
promoting stable junctions between rotating and non-rotating cells. Consistent
with this hypothesis, levels of Ed, an Egfr inhibitor, are very low in
ommatidial cells both when rotation commences and during the fast phase of
rotation (Fetting, 2009).
Early in the second half of rotation, it is proposed that higher levels of Ed
activity are necessary to repress Egfr signaling at the rotation interface,
possibly increasing the amount of active Cno and consequently increasing the
number of stable AJs between the moving and stationary cells. The more tightly
the cells adhere to one another, the less permissive the environment is for
movement, and the more difficult rotation becomes. Ed levels are high in the
cells in which it would need to be high, i.e., R1, R6, R7 and the cone cells.
Once rotation is complete, Ed and Fred are at high levels at the cell
boundaries between the interommatidial and ommatidial cells, an indication
that stable AJs now cement the fully rotated ommatidia in place (Fetting, 2009).
ed and fred interact genetically with the R3 and R4
genes, respectively, modifying only the degree-of-rotation aspect of the PCP
phenotype. Genetic and molecular epistasis data suggest that ed
and fred act in a pathway either downstream of, or parallel to, the
PCP genes. First, localization of Ed and Fred does not require the PCP
complex, nor do the PCP proteins require Ed and Fred for their localization.
Second, mutations in ed and fred affect only one aspect of
the PCP phenotype (Fetting, 2009).
Nectins and afadins have been implicated in numerous human diseases and
developmental defects, including breast cancer, metastasis and cleft palate.
Defective cell adhesion and cell signaling also underlie these problems. Given
the interspecies conservation of AJ genes, similar mechanisms might control
ommatidial rotation and contribute to these human diseases (Fetting, 2009).
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canoe:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
date revised: 20 December 2009
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