shotgun
See the embryonic expression pattern of shg at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
Maternal SHG mRNA is distributed uniformly in early embryos. During cellularization, SHG zygotic transcript is found in all blastoderm cells, but is excluded from cells of the presumptive mesoderm and endoderm that will convert into mesenchymal cells after gastrulation. Later in development, two mesodermally derived epithetial cell sheets express shg: the dorsal vessel and the gonadal sheet. The endoderm initiates zygotic shg expression just before its cells convert back into epithelial cells forming the lining of the larval midgut.
The amnioserosa [Images], the ectodermal epithelia and its epithelial derivatives (epidermis, tracheal system, foregut, hindgut, Malpighian tubules, and salivary glands) express shg continuously. shg is downregulated in neural precursors while they undergo an epithelial-mesenchymal transition and thereby segregate from the neurectodermal epithelium. shg is active however, in midline cells and the subperineurial glial sheath that forms the blood-brain barrier (Tepass, 1996).
Cadherin-N mRNA is first seen within nuclei of presumptive mesodermal cells prior to gastrulation at stage 5. mRNA transport to the cytoplasm starts at about stage 6-7, and the messengers are distributed throughout the cytoplasm by stage 8. Cadherin-N protein first appears at intercellular contacts in the mesoderm at stage 9, and then the protein is detected at boundaries of mesodermally derived cells that inititate transcription of the Shotgun gene at stage 13. Cadherin-N also appears in developing neural cells, presumably at their postmitotic stage; subsequently, Cadherin-N accumulates in axons of the entire CNS. At the subcellular level, neuronal processes including growth cones are labeled. Gastrulation and neurulation coincide with a switch of cadherin expression from Shotgut to Cadherin-N. Glial cells do not express Cadherin-N (Iwai, 1997).
During Drosophila gastrulation, morphogenesis occurs as a series of cell shape changes and cell
movements that probably involve adhesive interactions between cells. The dynamic aspects of cadherin-based cell-cell adhesion were examined in the morphogenetic events to
assess the contribution of such activity to morphogenesis. Shotgun and Cadherin-N show complementary expression
patterns in the presumptive ectoderm and mesoderm at the mRNA level. Switching of
cadherin expression from the Shotgun to the CadN type in the mesodermal germ layer occurs downstream
of the mesoderm-determination genes twist and snail. In contrast to twi and sna mutations, folded gastrulation mutants show normal replacement of Shotgun with CadN in cells corresponding to the mesoderm. Shotgun mRNA is present uniformly in the embryo until late stage 5, but it begins to disappear in the presumptive mesoderm shortly before the onset of ventral furrow formation. After stage 7 Cadherin-N mRNA is visible in the mesoderm. However, examination of cadherin protein expression
patterns shows that considerable amounts of Shotgun remains on the surfaces of mesodermal
cells during invagination, while CadN does not appear on the cell surfaces at this stage. Further
immunocytochemical analysis of the localizations of Shotgun and its associated proteins Armadillo
(beta-catenin) and Dalpha-catenin reveals dynamic changes in their distributions that are
accompanied by changes in cell morphology in the neuroectoderm and mesoderm. Shotgun, together with Armadillo and Dalpha-catenin, most strongly accumulate at apical contacts of neuroectodermal cells, at the same time that large apical junctions (AJs) are observed at the corresponding sites. As soon as the germ band starts to elongate (stage 8), the apical accumulation along lateral cell surfaces becomes disordered or obscure. Adherens junctions, based on the cadherin-catenin system, change their location,
size, and morphology. At this time large AJs are rarely found. During mesodermal invagination, as invaginating mesodermal cells are converted from wedge-shaped to round cells, Shotgun is gradually redistributed from AJs to a uniform distribution over the entire cell surface, including the cell contact-free areas in rounded mesodermal cells at stage 8. After this stage, Shotgun is completely eliminated from the mesoderm, and Arm and Dalpha-catenin are reduced to undetectable levels. These dynamic aspects of cadherin-based cell-cell adhesion appear to be
associated with the following: (1) initial establishment of the blastoderm epithelium; (2) acquisition of
cell motility in the neuroectoderm; (3) cell sheet folding, and (4) epithelial to mesenchymal conversion
of the mesoderm. These observations suggest that the behavior of the Shotgun-catenin adhesion
system may be regulated in a stepwise manner during gastrulation to perform successive
cell-morphology conversions. Also discussed are the processes responsible for loss of epithelial cell polarity and
elimination of preexisting Shotgun-based epithelial junctions during early mesodermal
morphogenesis (Oda, 1998a).
Cells in vascular and other tubular networks require apical polarity in order to contact each other properly and to form lumen. As tracheal branches join together in Drosophila melanogaster embryos, specialized cells at the junction form a new E-cadherin-based contact and assemble an associated track of F-actin and the plakin Short stop (shot). In these fusion cells, the apical surface determinant Discs lost (Dlt: now redefined as Drosophila Patj) is subsequently deposited and new lumen forms along the track. In shot mutant embryos, the fusion cells fail to remodel the initial E-cadherin contact, to make an associated F-actin structure and to form lumenal connections between tracheal branches. Shot binding to F-actin and microtubules is required to rescue these defects. This finding has led to an investigation of whether other regulators of the F-actin cytoskeleton similarly affect apical cell surface remodeling and lumen formation. Expression of constitutively active RhoA in all tracheal cells mimics the shot phenotype and affects Shot localization in fusion cells. The dominant negative RhoA phenotype suggests that RhoA controls apical surface formation throughout the trachea. It is therefore proposed that in fusion cells, Shot may function downstream of RhoA to form E-cadherin-associated cytoskeletal structures that are necessary for apical determinant localization (Lee, 2002).
The tracheal lumen is initially closed at branch tips. Concurrent with branching morphogenesis, specialized cells at branch tips, known as fusion cells, join branches into a continuous tubular network. This process of anastomosis requires each fusion cell to recognize its partner in the adjacent hemisegment and to form a lumen that connects the two branches. Shotgun, the Drosophila homolog of the cell adhesion molecule E-cadherin is integral to the initial fusion cell contact. Mutations in shotgun affect tracheal branch extension and lumen formation at anastomosis sites, as do mutations in armadillo, the Drosophila homolog of its effector ß-catenin. E-cadherin and ß-catenin control cell polarity and tube extension in culture, suggesting an evolutionarily conserved role for cadherin-mediated cell adhesion in apical surface regulation (Lee, 2002).
The results presented here provide further insights into how the cytoskeleton and associated proteins support contact formation and subsequent apical surface remodeling. The F-actin- and microtubule-binding domains of Shot are required to maintain and remodel E-cadherin contacts and to assemble a track of F-actin and Shot in fusion cells. This track initiates at the E-cadherin contact and extends outwards from it to connect with the existing apical assemblies of F-actin and Shot. It is proposed that the track guides new apical surface formation. Apical surface determinants and membrane appear to accumulate along the track, possibly by spreading from existing apical concentrations. This track may also enable the fusion cells to contract and to draw the existing lumenal surfaces closer, as fusion cells appear notably less compact in shot mutant embryos (Lee, 2002).
shot is required in neurons for growth cone motility. shot is also required to remodel E-cadherin-containing contacts between tracheal fusion cells. Surprisingly, Shot proteins perform these distinct morphogenetic roles using different combinations of the same cytoskeletal interaction domains. In fusion cells, the binding sites for F-actin and microtubules appear functionally redundant. The F-actin binding domain is essential when the GAS2 microtubule binding site is absent, and the GAS2 microtubule binding site is essential when the F-actin binding site is absent. By contrast, during axon extension, the Shot behaves as an F-actin/microtubule cross-linker because the cytoskeletal interaction domains are both individually essential and required in the same molecule (Lee, 2002).
These observations suggest that direct interactions between Shot and cytoskeletal proteins organize the cytoskeleton in fusion cells. The F-actin and microtubule domains may directly enable the accumulation of their cytoskeletal partners at the E-cadherin contact. In support of this hypothesis, the structurally similar F-actin binding domain of plectin alters F-actin organization and the GAS2 motif stabilizes associated microtubules against depolymerization in cultured cells. Since Shot’s interactions either with F-actin or with microtubules suffice to organize both cytoskeletal elements, binding to either F-actin or microtubules may then enhance other organizing interactions between F-actin and microtubules (Lee, 2002).
These other interactions may involve molecules required for E-cadherin signaling. E-cadherins are physically linked to F-actin via the ß-catenin/alpha-catenin complex and to dynein, a microtubule-based motor, via ß-catenin. They can further regulate actin dynamics via association with p120, a RhoA antagonist; E-cadherins also stabilize microtubule minus ends in cultured cells. E-cadherin signaling may therefore affect other proteins mediating interactions between F-actin and microtubules. Candidates include other F-actin/microtubule cross-linkers, regulators of Rho family GTPases that bind to microtubules and F-actin-based motors that form complexes with microtubule-based motors. Further analysis will be necessary to identify these other molecules in fusion cells: these other cytoskeletal regulators may permit residual anastomoses in shot mutant embryos (Lee, 2002).
In cells throughout the trachea, reduced RhoA activity disrupts lumen formation and partially disrupts Dlt (now Patj) localization. Tracheal expression of RhoAN19 does not appreciably affect E-cadherin localization. In cultured epithelial cells, E-cadherin localization is also resistant to RhoAN19. These findings are consistent with RhoA functioning downstream of or parallel to E-cadherin. E-cadherin-associated p120ctn negatively regulates RhoA, but whether a similar pathway operates in Drosophila is unknown (Lee, 2002).
In fusion cells, RhoA can also function upstream of E-cadherin, as constitutively active RhoAV14 affects E-cadherin localization selectively in these cells. E-cadherin distribution is more dynamic in fusion cells than in other tracheal cells, and may therefore be more sensitive to RhoAV14. RhoAV14 also affects new E-cadherin contacts in culture. Further experiments will reveal whether Shot, RhoA and E-cadherin function in a common, evolutionarily conserved pathway to regulate apical surface remodeling in fusion cells (Lee, 2002).
Cell rearrangement, accompanied by the rapid assembly and disassembly of cadherin-mediated cell adhesions, plays essential roles in epithelial morphogenesis. Various in vitro and cell culture studies on the small GTPase Rac have suggested it to be a key regulator of cell adhesion, but this notion needs to be verified in the context of embryonic development. The tracheal system of Drosophila was used to investigate the function of Rac in the epithelial cell rearrangement, with a special attention to its role in regulating epithelial cadherin activity. A reduced Rac activity leads to an expansion of cell junctions in the embryonic epidermis and tracheal epithelia, which was accompanied by an increase in the amount of Drosophila E-Cadherin-Catenin complexes by a post-transcriptional mechanism. Reduced Rac activity inhibits dynamic epithelial cell rearrangement. In contrast, hyperactivation of Rac inhibits assembly of newly synthesized E-Cadherin into cell junctions and causes loss of tracheal cell adhesion, resulting in cell detachment from the epithelia. Thus, in the context of Drosophila tracheal development, Rac activity must be maintained at a level necessary to balance the assembly and disassembly of E-Cadherin at cell junctions. Together with its role in cell motility, Rac regulates plasticity of cell adhesion and thus ensures smooth remodeling of epithelial sheets into tubules (Chihara, 2003).
Cadherin-based cell adhesions are vital to maintain the morphological and
functional features of the epithelium of multicellular organisms. During
morphogenesis of the epithelia, cell adhesions must be disrupted and
re-assembled in a regulated manner to allow movement of individual cells in
the epithelia. In vivo analyses have demonstrated that a reduction in Rac
activity prevents cell rearrangement. This phenotype is associated with an
increase in the level of E-Cadherin and its associated molecules, and
expansion of E-Cadherin localization to the basolateral membrane. It is inferred that increased E-Cadherin expression consolidates cell adhesiveness.
Hyperactivation of Rac prevents incorporation of newly synthesized E-Cadherin
into cell junctions and reduces cell adhesiveness, transforming the tracheal
epithelium into mesenchyme. It is suggested that switching of Rac between active
and inactive states promotes turnover of the complex containing E-Cadherin at
the cell junction, and maintains the plasticity of the tracheal epithelium to
allow branching morphogenesis (Chihara, 2003).
Expression of a dominant-negative form of Rac 1
greatly reduces cell rearrangement required for partitioning cells into the
stalk of the dorsal branch. Overproduction of this form, Rac 1N17, would shift the
cellular pool of Rac toward the inactive GDP-bound state. It is suggested that
turnover of E-Cadherin at a proper level requires a high level of Rac
activity. However, since highly active movement of cell extensions in the cells at
the tip is still visible, it is suggested that the ability of those cells to move
toward their target is mostly intact. A stronger reduction in Rac activity
might be required to demonstrate a role for Rac in promoting cell extensions,
as proposed from studies on tissue culture cells (Chihara, 2003).
Time-lapse analysis demonstrates that reduced Rac activity inhibits
cell rearrangement during branching of tracheal tubules. Under this condition,
the amounts of cadherins and catenins were increased and filled the cell
membrane. This phenotype is different from the phenotype of E-Cadherin-GFP
overexpression, which does not inhibit cell rearrangement. It is
suggested that a reduction in Rac promotes the association of cadherin-catenin
complexes with the cell membrane and stabilization of these complexes.
Activation of Rac resulta in an opposite phenotype characterized by the loss
of E-Cadherin and cell dissociation, and in prevention of E-Cadherin-GFP from
accumulating at apical cell junctions. All of these observations are
consistent with a hypothesis that Rac regulates the formation of
cadherin-catenin complexes at the cell junction. Incorporation of a
cadherin-catenin complex into the cellular junction would explain
stabilization of the complex when Rac activity is reduced. Possible modes of
Rac action on cadherin include apical transport and assembly/stabilization of
the complex. It is suggested that the inhibitory action on the cadherin cell
adhesion system is a general property of Rac in the Drosophila
embryo (Chihara, 2003).
How E-cadherin controls the elaboration of adherens junction-associated cytoskeletal structures crucial for assembling tubular networks was investigated. During Drosophila development, tracheal branches are joined at branch tips through lumens that traverse doughnut-shaped fusion cells. Fusion cells form E-cadherin contacts associated with a track that contains F-actin, microtubules, and Short stop (Shot), a plakin that binds F-actin and microtubules. Live imaging reveals that fusion occurs as the fusion cell apical surfaces meet after invaginating along the track. Initial track assembly requires E-cadherin binding to ß-catenin. Surprisingly, E-cadherin also controls track maturation via a juxtamembrane site in the cytoplasmic domain. Fusion cells expressing an E-cadherin mutant in this site form incomplete tracks that contain F-actin and Shot, but lack microtubules. These results indicate that E-cadherin controls track initiation and maturation using distinct, evolutionarily conserved signals to F-actin and microtubules, and employs Shot to promote adherens junction-associated cytoskeletal assembly (Lee, 2003).
Junctional contacts between cells are important for organizing the
cytoskeleton and regulating cell polarity. The large size
of plakins and their modular abilities to bind different cytoskeletal elements
make them potentially well suited to play key organizational roles.
However, except in the case of desmosomes, where the plakin desmoplakin
appears to be a crucial for organizing junction-associated cytoskeleton,
functional association of plakins with other cell-cell junctions has not been
described (Lee, 2003).
In selected cell types, Shot localizes with proteins of
the adherens junction and may play a role in adherens junction-mediated
organization of the cytoskeleton. It is proposed that Shot and E-cadherin form a
feedback loop in which E-cadherin, via ß-catenin, recruits Shot to new
contacts between the fusion cells and Shot stabilizes the contacts. The
cytoskeleton organizes around these contacts because adherens junction
associated Shot promotes the assembly of an F-actin/microtubule-rich track.
This track grows to span the fusion cells, extending the reach of the
junctions through the cells. The recruitment mechanism may be indirect in that
new adherens junctions in fusion cells are centers for cytoskeletal assembly,
and Short Stop binds F-actin and microtubules. Alternatively, Shot may
associate directly with E-cadherin or associated proteins. The assembly of
Shot with F-actin and microtubules may stabilize E-cadherin contacts simply by
bringing in cytoskeletal proteins that bind E-cadherin or associated proteins.
For example, EB1, which is present in the fusion track, co-immunoprecipitates
with a C-terminal fragment of Shot in cultured cells and associates with APC. APC interacts with ß-catenin to control tubulogenesis in vitro (Lee, 2003).
It is proposed that the assembly and maturation of a cytoskeletal intermediate
are two E-cadherin-dependent steps in tracheal cell fusion. Imaging of fixed and
live embryos suggests that fusion proceeds through the assembly and maturation
of a cytoskeletal track associated with adherens junctions. The track forms
after contact between the fusion cells, and persists for ~1 hour before
fusion occurs (Lee, 2003).
In this model, the ß-catenin-binding site and the juxtamembrane site
in the E-cadherin cytoplasmic domain operate sequentially and in the same
E-cadherin molecule to promote fusion. In mutant embryos in which either
ß-catenin or its binding site is defective, fusion cells make contact but
track assembly is not observed. These data suggest that E-cadherin may
initiate track assembly via ß-catenin. A mutation in the juxtamembrane
site dominantly inhibits track maturation. Microtubules are generally absent
from fusion tracks in these embryos, though some F-actin and Shot assembly
occurs. In E-cadherin/shotgun (shg) mutant embryos,
E-cadherin bearing this juxtamembrane mutation supports a low level of
F-actin/Shot track formation, but the tracks do not mature. In addition, this
juxtamembrane mutant E-cadherin causes progressive delocalization of the
apical tracheal cytoskeleton in shg mutant embryos (Lee, 2003).
Both the ß-catenin and juxtamembrane binding sites are required for
E-cadherin localization to adherens junctions, although only the juxtamembrane
mutation seems to interfere with endogeneous E-cadherin localization. The
results suggest that like mammalian E-cadherin, an evolutionarily conserved
juxtamembrane site is required for some E-cadherin functions. Similar effects
of mutations in the juxtamembrane site were observed in mammalian tissue
culture cells. However, juxtamembrane site function in Drosophila
E-cadherin probably does not require p120 (Lee, 2003).
Dominant effects on localization appear sensitive to expression levels,
whereas effects on fusion are less so, suggesting that defects in localization
are not enough to explain the defects in track maturation. Possibly, effects
on localization also reflect defects in organizing the cytoskeleton, as has
been observed in studies in which dominant alleles of Rho family GTPases
affect cadherin localization in culture (Lee, 2003).
It is proposed that the ß-catenin-binding site and ß-catenin are
required for track assembly, and that the juxtamembrane site regulates other
proteins involved in a later maturation step. This later step
likely requires microtubules. The microtubules or associated proteins may
reinforce the initial F-actin assembly in the track, as F-actin in fusion
tracks appears to be abnormally or poorly assembled in embryos expressing
AAA-JXT mutant E-cadherin in tracheal cells. The microtubules appear to be also
required for remodeling the fusion cell apical surfaces and also for bringing
them together to fuse. In embryos expressing AAA-JXT mutant E-cadherin in
tracheal cells, fusion cell apical surfaces do not develop or seal gaps at
appropriate times, and fusion tracks persist substantially longer, if they
resolve at all (Lee, 2003).
The microtubule regulated steps during fusion therefore likely involve
effects on F-actin dynamics. Microtubule-associated factors that may regulate
the F-actin cytoskeleton include Rac GTPase and exchange factors for Rho GTPase. Rac1 affects E-cadherin dependent adhesion in tracheal cells and a mutation in the juxtamembrane site in mammalian E-cadherin analogous to the one described in this study affects Rac activation. RhoA activation inhibits fusion track assembly.
Downstream interactions between F-actin and microtubules, such as those
mediated by Shot, may vary with cell type to produce distinct morphogenetic
outcomes. Further studies of tracheal tube fusion, a genetic system in which
adherens junction associated structures can be visualized in living embryos,
promises to identify the regulatory molecules that allow E-cadherin to direct
F-actin and microtubule assembly from the ß-catenin binding and
juxtamembrane domains (Lee, 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).
Epithelial cell migration and morphogenesis require dynamic remodeling of the actin cytoskeleton and cell-cell adhesion complexes. Numerous studies in cell culture and in model organisms have demonstrated the small GTPase Rac to be a critical regulator of these processes; however, little is known about Rac function in the morphogenic movements that drive epithelial tube formation. This study used the embryonic salivary glands of Drosophila to understand the role of Rac in epithelial tube morphogenesis. Inhibition of Rac function, either through loss of function mutations or dominant-negative mutations, disrupts salivary gland invagination and posterior migration. In contrast, constitutive activation of Rac induces motile behavior and subsequent cell death. Rac regulation of salivary gland morphogenesis occurs through modulation of cell-cell adhesion mediated by the E-cadherin/ß-catenin complex, and shibire, the Drosophila homolog of dynamin, functions downstream of Rac in regulating ß-catenin localization during gland morphogenesis. These results demonstrate that regulation of cadherin-based adherens junctions by Rac is critical for salivary gland morphogenesis and that this regulation occurs through dynamin-mediated endocytosis (Pirraglia, 2006).
This study shows that the Rac GTPases regulate salivary gland morphogenesis through modulation of cadherin/catenin-based cell–cell adhesion, likely by dynamin-mediated endocytosis. The characterization of the Rac mutant phenotypes suggests a model where Rac normally regulates cadherin-mediated cell–cell adhesion in salivary gland cells to allow enough plasticity for its invagination and migration yet keep the cells of the tube adhered to one another so that the gland can migrate as a cohesive tube. One mechanism by which cell surface cadherin levels are regulated is through selective endocytosis of E-cadherin from the apical–lateral membrane in a dynamin-mediated process. When Rac function is compromised through loss-of-function mutations or expression of dominant-negative mutations, the balance between E-cadherin at the plasma membrane and internalized E-cadherin appears to be abrogated so that more E-cadherin remains at the plasma membrane resulting in increased cell–cell adhesion and causing the gland to sever. These studies reveal the importance of precise regulation of adherens junction remodeling during cell migration in the context of a developing organ (Pirraglia, 2006).
In all stage 14 Rac1L89 mutant embryos examined, the salivary gland broke apart close to its approximate mid-point. Reduction in cadherin levels rescues the mutant Rac severing phenotype, suggesting that severing occurs because loss of Rac leads to an increase in cadherin-mediated cell–cell adhesion. At least two possible explanations for the midpoint severing phenotype are envisioned. In the first scenario, levels of cadherin remodeling may differ throughout the gland such that in Rac1L89 embryos the cells in the distal tip are least affected and cells in the mid-region of the gland are most affected by the increase in cadherin function. In this situation, when the distal cells begin to migrate posteriorly, the increased adhesivity of the mid-region cells prevents their migration and causes the gland to sever in the middle. In the second scenario, movement of the mid-region and the distal region of the gland may occur through different mechanisms. It is possible that while the distal most cells migrate by undergoing cell shape change and extending prominent protrusions in the direction of migration, cells in the middle of the gland may follow the distal cells by rearranging their positions along the gland, such as occurs during the convergence extension movements observed in epithelial morphogenesis. Dynamic remodeling of E-cadherin may be particularly important for proper rearrangement of the mid-region cells and an inability to rearrange when E-cadherin adhesion is increased may cause severing of the gland and subsequent separation of the migrating distal portion from the rest of the gland. Alternatively, it is possible that both of these scenarios are at play during normal salivary gland migration. Currently it is not possible to distinguish between these possibilities. In the developing tracheal tubes, Rac1 is required for cell rearrangements; in tracheal cells expressing a dominant-negative Rac1 mutation, the dorsal branch was shorter than that of wild-type embryos. Therefore, it will be important to determine whether cell rearrangement plays a role during salivary gland migration and to further elucidate the role of the Rac genes in this process (Pirraglia, 2006).
When Rac1 function is over-activate, dynamin-mediated endocytosis of E-cadherin may be increased, resulting in decreased cadherin at the plasma membrane, and decreased cell–cell adhesion. The loss of adhesion leads to the dispersal of salivary gland cells and ultimately cell death. Preventing Rac1V12-induced cell death led to the formation of abnormally shaped glands demonstrating that the Rac1V12 salivary gland phenotype is primarily due to abrogation of gland morphogenesis and not to activation of the apoptotic pathway. Moreover, since wild-type full-length E-cadherin is sufficient to rescue the Rac1V12 salivary gland phenotype, loss of cadherin function appears to be the primary cause for salivary gland defects. Thus, the Rac genes function in salivary gland cells to regulate E-cadherin-mediated cell–cell adhesion during tube morphogenesis (Pirraglia, 2006).
During salivary gland morphogenesis, gland integrity is kept intact while cells perform extensive cell shape changes and movements. Rac-regulated endocytosis of E-cadherin is one mechanism by which cell–cell adhesion is likely to be downregulated temporarily. After E-cadherin is endocytosed, it can be recycled back to the cell surface, sequestered transiently inside the cell or routed to late endosomes and lysosomes for degradation. Once salivary gland migration is complete and the gland has reached its final position, cell–cell adhesion may then need to be strengthened again in the mature gland and Rac activity may be downregulated to promote increase in surface cadherins (Pirraglia, 2006).
In addition to endocytosis, studies in mammalian cultured cells have shown that Rac can regulate levels of cell surface E-cadherin by other mechanisms, such as cleavage by presenilins and metalloproteinases, or tyrosine phosphorylation of the cadherin adhesion complex in a process involving reactive oxygen species. Thus, it will be interesting to determine whether additional mechanisms of E-cadherin regulation exist in salivary gland cells during gland morphogenesis (Pirraglia, 2006).
Numerous studies in cell culture have demonstrated that recycling of E-cadherin occurs in both a clathrin-dependent and caveolin-dependent manner. Since dynamin mediates both clathrin- and caveolin-dependent endocytosis, these studies do not allow distinguishing which type is involved in cadherin endocytosis during salivary gland migration. Alternatively, both types of endocytosis may mediate Rac1 regulation of E-cadherin in salivary gland cells (Pirraglia, 2006).
Expression of the Rac1V12 mutation in salivary gland cells leads to loss of expression of salivary gland specific proteins, apical–basal polarity proteins and E-cadherin/β-catenin. Concomitant with changes in gene expression, Rac1V12 mutant salivary gland cells lose adhesion to each other and subsequently migrate away or die by apoptosis. The data suggest that overactivation of Rac1 primarily affects E-cadherin/β-catenin-mediated adhesion and salivary gland cell fate and that the observed cell death is a secondary consequence of these earlier changes. When cell death was prevented in Rac1V12 embryos by expressing p35, more cells expressed the salivary gland specific protein PH4αSG1 than in Rac1V12 embryos; however, the expression level was drastically reduced compared to wild-type, suggesting that even in the Rac1V12p35 cells, cell differentiation was still mostly altered. Moreover, Rac1V12p35 salivary gland cells did not form a normal gland, demonstrating a role for Rac1 in gland morphogenesis. It is possible that apoptosis of Rac1V12 cells is brought about by the loss or reduction of Forkhead (Fkh) function. Fkh is expressed early in the salivary gland placode and its expression is maintained throughout embryogenesis. In the absence of fkh function, salivary gland cells die by apoptosis during the invagination stage. Since expression of dCREB-A and PH4αSG1 is reduced in Rac1V12 mutant salivary gland cells, it is possible that Fkh expression is also similarly reduced, thereby, causing the cells to undergo apoptotic cell death (Pirraglia, 2006).
Many human cancers are due to epithelial-derived tumors. When epithelial cells metastasize, they first undergo an epithelial to mesenchymal transition (EMT) before migrating away from the primary tumor to invade surrounding tissues. EMT is characterized by the loss of epithelial polarity and cell–cell adhesion. When Rac1V12 was expressed in salivary gland cells, expression of apical membrane proteins, Crumbs and aPKC and adherens junction proteins E-cadherin and β-catenin, was either lost or mislocalized. Based on these criteria, activation of Rac1 function induces features characteristic of early changes in EMT and metastasis. Interestingly, the expression levels of Rho GTPases are found to be elevated in a number of human cancers. For example, increased Rac protein levels and fast-cycling Rac mutations have been correlated with colorectal and breast tumors. Expression of constitutively active Rac1 causes some salivary gland cells to lose polarity and adhesion to neighboring cells and migrate away in a manner similar to EMT. These findings suggest that Rac1-regulated endocytosis of E-cadherin in the Drosophila salivary glands may be critical in maintaining epithelial character and preventing the loss of cell–cell adhesion and cell polarity. The Drosophila salivary gland might thus be powerful as a simple system to identify and characterize mechanisms that regulate cadherin-based cell–cell adhesion and certain aspects of EMT (Pirraglia, 2006).
Epithelial tissues maintain a robust architecture during development. This fundamental property relies on intercellular adhesion through the formation of adherens junctions containing E-cadherin molecules. Localization of E-cadherin is stabilized through a pathway involving the recruitment of actin filaments by E-cadherin. This study identifies an additional pathway that organizes actin filaments in the apical junctional region (AJR) where adherens junctions form in embryonic epithelia. This pathway is controlled by Bitesize (Btsz), a synaptotagmin-like protein that is recruited in the AJR independently of E-cadherin and is required for epithelial stability in Drosophila embryos. On loss of btsz, E-cadherin is recruited normally to the AJR, but is not stabilized properly and actin filaments fail to form a stable continuous network. In the absence of E-cadherin, actin filaments are stable for a longer time than they are in btsz mutants. Two polarized cues have been identified that localize Btsz: phosphatidylinositol (4,5)-bisphosphate, to which Btsz binds; and Par-3. Btsz binds to the Ezrin–Radixin–Moesin protein Moesin, an F-actin-binding protein that is localized apically and is recruited in the AJR in a btsz-dependent manner. Expression of a dominant-negative form of Ezrin that does not bind F-actin phenocopies the loss of btsz. Thus, these data indicate that, through their interaction, Btsz and Moesin may mediate the proper organization of actin in a local domain, which in turn stabilizes E-cadherin. These results provide a mechanism for the spatial order of actin organization underlying junction stabilization in primary embryonic epithelia (Pilot, 2006).
Homotypic binding of the cell-adhesion molecule E-cadherin (E-cad) at the adherens junctions of epithelial cells organizes the formation of multiprotein complexes, composed in part of the ß-catenin and alpha-catenin proteins, and their dynamic interaction with actin filaments (F-actin). F-actin is required to stabilize E-cad–ß-catenin–alpha-catenin complexes. Moreover, E-cad regulates its own stability through the organization of actin filaments through alpha-catenin: alpha-catenin binds Formin (also known as Diaphanous) and suppresses branching by competing with Arp2/3 (Drees, 2005). When epithelia form through the mesenchymal–epithelial transition, the sites of initial cell contact serve as spatial landmarks for the recruitment of E-cad–ß-catenin–alpha-catenin complexes during the formation of adherens junctions. In primary embryonic epithelia, however, adherens junctions do not form through specific cell contacts, and the spatial cues positioning the adherens junctions in the AJR are less characterized and may be different. The identification of such spatial cues and the mechanisms whereby these cues organize structural, cytoskeletal components associated with the formation and/or stabilization of adherens junctions is an important challenge (Pilot, 2006).
This problem was addressed in the early Drosophila embryo. Formation, stabilization and remodelling of adherens junctions occur in a tightly and genetically controlled sequence involving e-cad (or shotgun), armadillo (or ß-catenin), par-6 , par-3 (or bazooka, baz), aPKC, crumbs and others. A microarray-based RNA interference (RNAi) screen of epithelial morphogenesis identified btsz, a gene previously known to control growth in adult flies (Serano, 2003), as a regulator of embryonic epithelial integrity. In embryos injected with double-stranded RNA (dsRNA) probes specific for btsz (hereafter called btszRNAi embryos), gastrulation is severely affected and the epithelium fails to extend properly. Defects are either strong or medium; that is, they are visible at the beginning of gastrulation or about 15 min later, respectively. The defects are penetrant (80%) and dose dependent. Four different, nonoverlapping probes produce these defects and embryos were not affected with control probes (Pilot, 2006).
Btsz is the only Drosophila member of the synaptotagmin-like protein (SLP) family characterized by the presence of tandem carboxy-terminal C2 boxes. btsz encodes several isoforms (Serano, 2003). In early embryos, btsz1 was not detected but btsz2 and btsz3 are expressed together with btsz0, another isoform not previously reported. At least one of these isoforms is maternally and zygotically provided. The most efficient dsRNA probes used recognizes all three maternally and zygotically expressed isoforms. These isoforms were strongly reduced in btszRNAi embryos, suggesting that RNAi produces a severe btsz loss-of-function phenotype (Pilot, 2006).
Two btsz alleles have been described (Serano, 2003): btszK13-4 introduces a deletion in the amino terminus of btsz2 (residues 501–1,494), btszJ5-2 corresponds to a frameshift mutation that introduces a stop codon at amino acid 390, which leads to a truncation in Btsz0 and Btsz2, and probably the absence of Btsz3. btszK13-4 homozygous female escapers can be recovered and were crossed to heterozygous btszK13-4 or btszJ5-2 males. Although many embryos were not fertilized, those that were reached cellularization and showed epithelial defects during gastrulation: 26% of btszK13-4/btszK13-4 and 46% of btszK13-4/btszJ5-2 embryos. btszJ5-2 germline clones do not produce eggs and btszJ5-2 is lethal. Trans-heterozygous embryos were examined with a deficiency removing the btsz locus (Df(3R)Exel6275, called Dfbtsz): 12% of embryos from crosses of Dfbtsz/btszK13-4 females and wild-type males showed epithelial defects. This proportion went up to 39% when males were heterozygous
btszK13-4/+. It is concluded that btsz is zygotically and maternally required. Whereas RNAi targeted all three btsz isoforms, btszK13-4 left intact a large fraction of Btsz2 and Btsz0, probably explaining the weaker penetrance of phenotypes in btszK13-4 (26%) or btszK13-4/btszJ5-2 (46%) mutants, as compared with btszRNAi embryos (80%). Notably, despite its essential role in the formation of epithelia in early embryos, the recovery of adult escapers suggests that btsz may be dispensable in adult epithelia (Pilot, 2006).
Overexpression of a btsz2 isoform lacking the 3' untranslated region (UTR) rescues the phenotypes produced by an RNAi probe targeting the 3' UTR of all btsz isoforms. Overexpression of btsz2 more robustly rescues the btszRNAi phenotype than does btsz3 overexpression, suggesting that btsz2 has a prominent role. The injection of morpholino antisense oligonucleotides (morpholinos) specific to each btsz isoform confirmed this: a control morpholino showed no defect, a mix of btsz0, btsz2 and btsz3 morpholinos caused penetrant defects (92%), and a btsz2-specific morpholino alone caused defects in 73% of embryos. Experimental focus was therefore placed on Btsz2, a 286-kDa protein (2,645 residues) (Pilot, 2006).
The expression of a Glu-epitope-tagged variant of Btsz2 (Btsz2–Glu) was strongly reduced in btszRNAi embryos. The epithelium failed to maintain its regular morphology in btszRNAi embryos, btsz mutants and btsz morphants. Although cellularization proceeds similarly in btszRNAi and control embryos, at the onset of gastrulation the epithelium collapses and becomes multilayered in btszRNAi and btszK13-4/btszJ5-2 mutant embryos, as compared with controls. A similar phenotype was observed in e-cadRNAi embryos. Thus, btsz controls the stable architecture of primary embryonic epithelia (Pilot, 2006).
These data suggested that btsz might regulate the formation of adherens junctions. In contrast to the wild type, in which E-cad is uniformly present at the adherens junctions, E-cad expression is heterogeneous and the adherens junctions appears severely fragmented in btsz mutants and btszRNAi embryos. Time-lapse recordings of E-cad fused to green fluorescent protein (GFP) showed that adherens junctions forms properly in the AJR of btszRNAi embryos but that, subsequently, E-cad–GFP expression disappears, suggesting a defect in the stabilization but not targeting of E-cad. E-cad–GFP, or endogenous E-cad, disappears in small patches at cell contacts, pointing to defects in actin organization. Indeed, the actin belt in the AJR is fragmented in btszRNAi embryos. Tested were performed to see whether actin organization or the E-cad–ß-catenin–alpha-catenin complexes was the primary cause of the disassembly of adherens junctions in btszRNAi embryos. In e-cadRNAi embryos, in which E-cad was undetectable in the nascent AJR, the actin belt is not considerably affected during early gastrulation and clearly less affected than in btszRNAi embryos at the same stage. Subsequently, however, F-actin was disorganized in e-cadRNAi embryos. This suggests that Btsz is part of an E-cad-independent pathway controlling actin organization in the AJR and consequently junction stability (Pilot, 2006).
Next, Btsz2 localization was examined. Btsz2–Glu is a functional protein that rescues the btszRNAi phenotype. Btsz2–Glu was previously reported to localize apically in follicular epithelial cells (Serano, 2003). In early embryos, Btsz2–Glu is detected at the AJR together with E-cad from the end of cellularization until about 30 min into gastrulation. Subsequently, Btsz2–Glu was found in a subapical compartment. At these early stages, E-cad colocalizes with Par-3 (also known as Baz). Therefore the possible role of E-cad and Par-3/Baz in Btsz2 localization in the AJR was addressed. In e-cadRNAi embryos, the recruitment of E-cad in the AJR is blocked and Btsz2 is normal; by contrast, in par-3/bazRNAi embryos Btsz2 is largely cytoplasmic, like PatJ, another marker of AJR at this stage. Btsz2 is thus a target of the early polarity marker Par-3/Baz, which is required for E-cad localization in the AJR (Pilot, 2006).
The role of the two C2 boxes (C2AB) in the localization of Btsz2 was tested. Purified glutathione S-transferase (GST)-tagged C2AB binds to phosphatidylinositol mono- and bisphosphate species in a Ca2+-dependent fashion in vitro. The in vivo relevance of this binding was assessed. A tagged form of Btsz2 lacking the C2 boxes (Btsz2-DeltaC2–HA) expressed in gastrulating embryos was cytoplasmic and failed to localize at the AJR. Conversely, an epitope-tagged form of C2AB (C2AB–HA) localizes at the plasma membrane in gastrulating embryos. Notably, C2AB is polarized and concentrates in the apical surface and in the AJR. Of all the phosphoinositides that C2AB binds in vitro, phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P2) is the most abundant at the plasma membrane, suggesting that PtdIns(4,5)P2 could be required for Btsz2 localization in the AJR. Injection of cellularizing embryos with neomycin, a compound that binds and inhibits PtdIns(4,5)P2, resulted in epithelial defects similar to btsz, par-3 or e-cadRNAi, and inhibits the recruitment of Btsz2 at the plasma membrane and the AJR. A fusion between GFP and the pleckstrin homology (PH) domain of phospholipase Cdelta (PLCdelta), which specifically binds PtdIns(4,5)P2, localizes apically and in the AJR, similar to the Btsz C2 boxes. It is concluded that PtdIns(4,5)P2 is a polarized spatial cue required for localization of Btsz in the AJR, and hence for adherens junction stability, together with Par-3/Baz (Pilot, 2006).
How does localized Btsz organize F-actin in the AJR? A large-scale two-hybrid screen identified an interaction between Btsz and Moesin, the only Drosophila member of the Ezrin–Radixin–Moesin (ERM) family of F-actin binding proteins that has been implicated in various aspects of epithelial polarity. This interaction was confirmed and Btsz was shown to bind to the third F3 subdomain of Moesin. A minimal region in Btsz that binds Moesin was narrowed down. This interaction occurred in GST pull-down assays of Drosophila S2 cell lysates and embryonic extracts. The functional relevance of this interaction was assessed. Moesin and the phosphorylated active form of Moesin, which binds F-actin, localizes in early embryos in the apical surface and in the AJR, together with Btsz2 and E-cad, suggesting that the interaction between Btsz and Moesin may spatially define a domain of actin organization in the AJR required to stabilize E-cad. In agreement with this, in btszRNAi and btsz mutant embryos, Moesin localization was diminished in the AJR as compared with controls, and E-cad and Moesin segregated in distinct domains as E-cad progressively disappeared (Pilot, 2006).
Whether Moesin is required for epithelial stability was tested in early embryos. Moesin has a major maternal contribution and is a very stable protein. Moreover, females whose germline is mutant for moesin do not lay eggs. Thus, no phenotype was identified using either various moesin mutant alleles or RNAi. Therefore a dominant-negative construct of Ezrin, a mammalian Moesin orthologue that lacks the C-terminal actin-binding domain and acts as a dominant-negative in Drosophila (EzrinDN, containing residues 1–280) was expressed in early embryos . Embryos expressing EzrinDN during gastrulation showed epithelial defects (41% of embryos) similar to btsz mutants. In particular, cellularization was normal, adherens junctions formed properly, but E-cad was no longer present homogeneously around the AJR (Pilot, 2006).
These results shed light on the mechanisms underlying the spatial control of actin filament and the stability of the adherens junctions in the Drosophila primary embryonic epithelium. In Btsz, an E-cad independent pathway has been identified that participates in F-actin organization in the AJR, together with Moesin. Btsz and Moesin bind and colocalize in the AJR in a btsz-dependent fashion, and expression of a mutant form of Ezrin that does not bind F-actin disrupts adherens junctions stability similar to loss of btsz. Notably, this work identifies upstream polarity cues (Par-3/Baz and PtdIns(4,5)P2) that control the spatial order of actin organization at the AJR through the localization of Btsz. The fact that PtdIns(4,5)P2 acts as a key regulator of epithelial polarity in the early embryo raises the issue of how PtdIns(4,5)P2 metabolism is spatially regulated in epithelial cells. The observation that Par-3 binds PTEN, which converts PtdIns(3,4,5)P3 into PtdIns(4,5)P2, suggests that Par-3/Baz may be part of this process. Thus a key intermediate between polarity cues and structural elements of adherens junctions important for embryonic epithelial stability has been identified. Five SLPs and two SLP-related (Slac2) proteins are close orthologues of Btsz in mammals. It would be worth investigating their potentially conserved role in the dynamic organization of actin at adherens junctions in embryonic epithelia (Pilot, 2006).
A fundamental requirement during organogenesis is to preserve tissue integrity to render a mature and functional structure. Many epithelial organs, such as the branched tubular structures, undergo a tremendous process of tissue remodelling to attain their final pattern. The cohesive properties of these tissues need to be finely regulated to promote adhesion yet allow flexibility during extensive tissue remodelling. This study reports a new role for the Egfr pathway in maintaining epithelial integrity during tracheal development in Drosophila. The integrity-promoting Egfr function is transduced by the ERK-type MAPK pathway, but does not require the downstream transcription factor Pointed. Compromising Egfr signalling, by downregulating different elements of the pathway or by overexpressing the Mkp3 negative regulator, leads to loss of tube integrity, whereas upregulation of the pathway results in increased tissue stiffness. Regulation of MAPK pathway activity by Breathless signalling does not impinge on tissue integrity. Egfr effects on tissue integrity correlate with differences in the accumulation of markers for cadherin-based cell-cell adhesion. Accordingly, downregulation of cadherin-based cell-cell adhesion gives rise to tracheal integrity defects. These results suggest that the Egfr pathway regulates maintenance of tissue integrity, at least in part, through the modulation of cell adhesion. This finding establishes a link between a developmental pathway governing tracheal formation and cell adhesiveness (Cela, 2006).
This study documents a new role for the Egfr pathway in the regulation of
tissue integrity. This new requirement could depend on the described early
peak of Egfr activity, which would be sufficient to prevent defects at later
stages. However, it is proposed that Egfr-promoted epithelial integrity
depends on a later, or continuous but lower, or basal activity of the pathway
that does not correlate with detectable ERK phosphorylation. Consistent with
this hypothesis, it was found that downregulation of the pathway by overexpressing
801 or UAS-EgfrDN with btlGal4, which is
expressed after the early peak of ERK phosphorylation, produces a conspicuous
branch integrity phenotype. In any case, tissue integrity defects are mainly
observed in the most dorsal and ventral tracheal branches, which are subjected
to stronger pulling forces as development proceeds, and, therefore, it is
precisely at late stages when defects in tissue integrity are expected (Cela, 2006).
AJs connecting epithelial cells dynamically disassemble and reassemble,
thereby allowing tissue remodelling. Tracheal tissue remodelling might require
the fine-tuning of cell adhesion properties, since tracheal cells need to be able
to change their relative position (probably by loosening cell adhesion) while
maintaining epithelial continuity. The data indicates that the Egfr pathway is
a modulator of this balance, not only in the tracheal system, but also in
other tissues undergoing extensive remodelling, such as the salivary glands,
where a similar regulation of DE-cad and actin levels is found upon modulation
of Egfr signalling. Conversely, no such a regulation was found in more static tissues, like the ectoderm, whose maintenance was proposed to depend on the maternally provided DE-cad protein. It is suggested that the Egfr pathway plays a role in the modulation of cell adhesion in tissues that undergo dramatic morphogenetic events, which might need the zygotic DE-cad contribution and a more dynamic regulation of cell adhesion. The results indicating a modulation of junctional complexes and/or the actin cytoskeleton by the Egfr pathway establish a link between a developmental pathway required for many biological events and cell
biology in terms of cell adhesiveness and cell shape (Cela, 2006).
The results show that downregulation of several intracellular elements of
the MAPK pathway produce defects in branch integrity, whereas a constitutively
activated form of rl (rlsem) rescues the
phenotype of btlGal4 801 embryos. This suggests that the conserved
MAPK cassette is required to maintain branch integrity (Cela, 2006).
Two tyrosine kinase receptors, Egfr and Btl, activate the MAPK pathway
during embryonic tracheal development. However, the two
receptors, acting through the same intracellular cascade, elicit different
responses. The MAPK pathway requirement in primary branching is likely to
depend on input by btl, whereas the tissue integrity requirement is
likely to depend on input by Egfr. How does the same MAPK pathway
trigger distinct outcomes depending on the receptor that activates it? A
temporal and/or spatial differential activation of the MAPK pathway could
account for the different outcome. In addition, differences in the composition
of the intracellular cascade due to specific transducers for one type of
receptor, such as downstream of FGFR (dof;
stumps-FlyBase), could contribute. Finally, quantitative and/or qualitative
differences in the activation of the intracellular transducers by the
different receptors could also underlie the outcome diversity (Cela, 2006).
Similar to these observations, air sac development in Drosophila has
been recently reported to require both Btl and Egfr, and each receptor seems
to elicit different responses. Furthermore, since during embryonic
tracheal development, an uncoupling of the MAPK cassette and pnt has
been observed during air sac development.
These parallels suggest a common mechanism for generating different responses
from the same intracellular transduction pathway (Cela, 2006).
The loss of tissue continuity and cell detachment observed in Egfr
downregulation conditions may be due, at least in part, to a decrease in cell
adhesion. Accordingly, a mild, but reproducible, decrease is observed in the
accumulation of DE-cad and cortical actin. As inferred from the phenotypes,
such a mild decrease could cause a loss of cell adhesion during tracheal
remodelling, while not grossly affecting other processes requiring
DE-cad-based cell adhesion, such as branch fusion. As expected, it was found that compromising AJ assembly or the
actin cytoskeleton also gives rise to defects in tracheal tissue
integrity (Cela, 2006).
Cadherins have been shown to support cell cohesion and participate in
morphogenetic events. The actin cytoskeleton also plays an important role in
shaping the cell architecture and in many morphogenetic processes. AJs and the
actin cytoskeleton are intimately coupled, and their formation and maintenance
is interdependent. Such interdependence is also observed in the tracheal
system (Cela, 2006).
Cadherin-based cell-cell adhesion can be regulated at transcriptional and
posttranscriptional levels. The modulation of a DE-cadGFP chimaera
driven by heterologous promoters shows that, in the current case, DE-cad regulation is posttranscriptional. Several posttranscriptional mechanisms of DE-cad
regulation have been proposed, and a role for the Egfr pathway can be envisaged in each of them. A first mechanism
is at the level of DE-cad endocytic trafficking. In this context, the Egfr
pathway could modulate the balance between recycling to the plasma membrane of
internalised DE-cad or lysosomal targetting and degradation. A second
mechanism of cell-cell adhesion regulation is posttranslational modifications
of AJ components, such as phosphorylation or ubiquitination. Finally, another
possible mechanism of regulation is through the cytoskeleton. The Rho family
of small GTPases plays a key role in actin cytoskeleton regulation, and growth
factor receptors such as Egfr have been reported to regulate their activity. Remarkably, the Egfr pathway has been recently shown to
regulate the expression of the rhoGAP cv-c in the tracheal placodes, and
it was found that cv-c mutants display tracheal integrity defects,
although they are milder than those seen upon downregulation of the Egfr
signal. It is therefore proposed that cv-c is at least one of the
effectors of Egfr-mediated modulation of DE-cad levels and tracheal tissue
integrity. Further analysis will be needed to disentangle the exact molecular
mechanisms and to find other possible mediators of the Egfr signal (Cela, 2006).
The decrease of cadherin activity upon activation of the Egfr pathway has
been extensively reported in the literature. This study reports the opposite: that Egfr pathway downregulation correlates with a decrease of cadherin-based cell adhesion. Although this is not the first example of such a relationship, it illustrates the versatility and complexity of the interactions occurring between signalling pathways and adhesion molecules, and establishes another model with which to analyse how cell adhesion is modulated (Cela, 2006).
The distribution of proteins has been analyzed in the apico-lateral cell junctions in Drosophila imaginal
discs. Antibodies to phosphotyrosine (PY),
Armadillo (Arm) and Drosophila E-cadherin (DE-cad) as well as FITC phalloidin marking filamentous
actin, label the site of the adherens junction, whereas antibodies to Discs large (Dlg), Fasciclin III
(FasIII) and Coracle (Cor) label the more basal septate junction. The junctional proteins labeled by
these antibodies undergo specific changes in distribution during the cell cycle. Previous work has shown that a loss-of-function dlg mutation, which causes neoplastic imaginal disc overgrowth, leads to
loss of the septate junctions and the formation of what appear to be ectopic adherens junctions . This study was extended to examine the effects of mutation in other genes that
also cause imaginal disc overgrowth. Based on staining with PY and Dlg antibodies, the apico-lateral
junctional complexes appear normal in tissue from the hyperplastic overgrowth mutants fat (coding for a novel cadherin) , discs overgrown, giant discs
and warts (coding for a homolog of human myotonic dystrophy kinase). However, imaginal disc tissue from the neoplastic overgrowth mutants dlg and lethal (2) giant larvae show
abnormal distribution of the junctional markers including a complete loss of apico-basal polarity in
loss-of-function dlg mutations. These results support the idea that some of the proteins of apico-lateral
junctions are required both for apico-basal cell polarity and for the signaling mechanisms controlling
cell proliferation, whereas others are required more specifically in cell-cell signaling (Woods, 1997).
In Drosophila, Src oncogene 1 was considered a unique ortholog of the vertebrate c-src; however, more recent evidence has been shown to the contrary. The
closest relative of vertebrate c-src found to date in Drosophila is not Dsrc64, but Dsrc41, a gene identified for the first time in this paper.
In contrast to Src64, overexpression of wild-type Src41 causes little or no appreciable phenotypic change in Drosophila.
Both gain-of-function and dominant-negative mutations of Src41 cause the formation of supernumerary R7-type neurons,
suppressible by one-dose reduction of boss, sevenless, Ras1, or other genes involved in the Sev pathway. Dominant-negative
mutant phenotypes are suppressed and enhanced, respectively, by increasing and decreasing the copy number of wild-type
Src41. The colocalization of Src41 protein, actin fibers and DE-cadherin, as well as the Src41-dependent disorganization of actin fibers
and putative adherens junctions in precluster cells, suggest that Src41 may be involved in the regulation of cytoskeleton
organization and cell-cell contacts in developing ommatidia (Takahashi, 1996).
Dynamically regulated cell adhesion plays an important role during animal morphogenesis. The formation of the visual system in Drosophila embryos has been used as a model system to investigate the function of the Drosophila classic cadherin, DE-cadherin, which is encoded by the shotgun (shg) gene. The visual system is derived from the optic placode, which normally invaginates from the surface ectoderm of the embryo and gives rise to two separate structures, the larval eye (Bolwig's organ) and the optic lobe. The optic placode dissociates and undergoes apoptotic cell death in the absence of Shotgun, whereas overexpression of Shotgun results in the failure of optic placode cells to invaginate and of Bolwig's organ precursors to separate from the placode. These findings indicate that dynamically regulated levels of Shotgun are essential for normal optic placode development. Overexpression of Shotgun can disrupt Wingless signaling through titration of Armadillo out of the cytoplasm to the membrane. However, the observed defects are likely the consequence of altered Shotgun mediated adhesion rather than a result of compromising Wingless signaling, since overexpression of a Shotgun-alpha-catenin fusion protein, which lacks Armadillo binding sites, causes defects similar to Shotgun overexpression. The genetic interaction between Shotgun and the Drosophila EGF receptor homolog, Egfr, was studied. If Egfr function is eliminated, optic placode defects resemble those following Shotgun overexpression, which suggests that loss of Egfr results in an increased adhesion of optic placode cells. An interaction between Egfr and Shotgun is further supported by the finding that expression of a constitutively active Egfr enhances the phenotype of a weak shg mutation, whereas a mutation in rhomboid (rho) (an activator of the Egfr ligand Spitz) partially suppresses the shg mutant phenotype. Finally, Egfr can be co-immunoprecipitated with anti-Shotgun and anti-Armadillo antibodies from embryonic protein extracts. It is proposed that Egfr signaling plays a role in morphogenesis by modulating cell adhesion (Dumstrei, 2002).
The head ectoderm of early Drosophila embryos is subdivided into several domains that realize different
morphogenetic programs. The embryonic eye field is the posterior-medial region of the procephalic neurectoderm that gives rise to the visual system, which includes the larval eye (Bolwig's organ) and adult
eye, as well as the optic lobe. Around gastrulation, cells of the eye field undergo a convergent extension directed laterally. Shortly afterwards these cells form two morphologically visible placodes, one on either side of the embryo. These optic placodes sink inside and become the optic lobe primordia, epithelial double layers attached to the posterior surface of the brain. The optic placode of a stage 12-13 embryo is V-shaped, with the anterior leg of the V representing the anterior lip, which later forms the inner anlage of the optic lobe, and the posterior leg forming the posterior lip, later forming the outer anlage. As the invagination deepens and the two lips 'sink' inside the embryo, ectodermal cells that earlier surrounded the perimeter of the optic placode approach each other and eventually form a
closed epidermal cover. Abundant cell death accompanies the closing of the head epidermis over the optic lobe anlage, and the subsequent separation of
this anlage from the epidermis. A small number of cells that originally formed part of the posterior lip of the optic placode
remain integrated in the head epidermis and form the larval eye or Bolwig's organ. As these cells move away from the optic lobe anlage their basal ends
become drawn out and form axons that constitute Bolwig's nerve (Dumstrei, 2002).
Shotgun is expressed throughout the ectoderm including the eye field and its epithelial derivatives. One would expect that normal optic
lobe development requires modulation of Shotgun activity to allow, for example, the segregation of the invaginating optic placodes from the
surrounding ectoderm. Since cell culture studies have indicated that the mammalian EGF receptor can disrupt cadherin-based adhesion, it was of interest to see whether Drosophila Egfr is expressed in the visual system to allow for such a possibility in Drosophila as well. Egfr is
expressed in a complex and dynamic pattern that closely parallels the pattern of double-phosphorylated ERK (dpERK) expression, indicating activation
of the MAP kinase signaling pathway. During stage 7 both rho (an activator of Egfr signaling) and dpERK are expressed in two stripes in the head ectoderm. The expression of dpERK in these two stripes is the result of Egfr activity. The anterior stripe corresponds to part of the head midline, while the posterior stripe reaches into the eye field. Distribution of dpERK in the two stripes becomes patchy during stage 10. At the
same time, the posterior stripe widens dorsally to overlap with part of the optic lobe placode. Finally, at the late extended germ band stage and
during germ band retraction, dpERK becomes restricted to the optic lobe placodes and cells of the dorsal head midline. This expression pattern demonstrates that Egfr activation accompanies the determination, morphogenesis and differentiation of the embryonic visual system (Dumstrei, 2002).
On the subcellular level, Egfr is expressed diffusely on the membrane of epithelial cells and neuroblasts. Egfr overlaps with Armadillo,
the Drosophila ß-catenin homolog, which is an integral component of the cadherin-catenin complex. Like Shotgun, Armadillo is
concentrated strongly in the apically located zonula adherens but is also found at lower levels in the entire lateral membranes (Dumstrei, 2002).
A second type of junction, called a septate junction, develops in Drosophila epithelial cells at a slightly later stage than the zonula adherens. Septate junctions have been implicated in maintaining epithelial stability. The Coracle protein forms part of
the septate junctional complex, and an antibody against Coracle serves as a sensitive marker for this junction. Applying this
marker to embryos of different stages it was found that all ectodermally derived epithelia express Coracle, except for the optic lobe and the invaginations
that form the stomatogastric nervous system. Accordingly, no septate junctions have been reported in previous electron microscopic
investigations of these tissues. The reliance on adherens junctions alone may make the optic lobe (and stomatogastric nervous system) susceptible to changes in the stability of these junctions; such changes occur resulting from manipulations of Shotgun and Egfr (Dumstrei, 2002).
A finely adjusted level of Shotgun is required for optic placode morphogenesis, and ß-catenin, as well as EGFR signaling, is involved in this process. Reduction in Shotgun results in dissociation of the placode around the time when it normally invaginates, suggesting that the forces exerted on the epithelial sheet while folding may disrupt cell contacts. A similar phenotype was described for other epithelial invaginations, including the Malpighian tubules and stomatogastric nervous system. Abolishing Armadillo/ß-catenin function results in a similar, if somewhat weaker phenotype. If Shotgun is overexpressed, invagination is also impaired. Cells stay together in a placode-like formation (as would be expected from 'hyperadhesive' epithelial cells), but do not noticeably constrict apically. It should be noted that the interpretation of this failure of optic placode cells to constrict is complicated by the accompanying increase in cell death in surrounding head epidermal cells. This phenomenon, in addition to a direct effect of an increased amount of Shotgun in the optic placode cells, could be part of the pathology responsible for the non-invagination phenotype. By contrast, the non-disjunction of optic lobe and larval eye is likely to be a rather direct consequence of an increased amount of Shotgun expression. Interestingly, other adhesion systems, notably the Drosophila N-CAM homolog FasII, are also involved in optic lobe-larval eye separation. Thus, the down regulation of FasII by the 'anti-adhesion' molecule Beaten path is also required for normal larval eye morphogenesis (Dumstrei, 2002).
Overexpression of Shotgun or the DE-cad-alpha-catenin fusion construct causes a dramatic change in optic lobe morphogenesis, without causing much disruption in other epithelia. It is speculated that this enhanced sensitivity of optic lobe cells towards an increased level of Shotgun 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, trachea and hindgut, septate junctions form by far the more prominent junctional complex. Septate junctions have been implicated in epithelial stability. 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. 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 (Dumstrei, 2002).
Expression of a fusion construct, DE-cad-alpha-catenin, in which the cytoplasmic domain of Shotgun is replaced by a truncated alpha-catenin, thereby preventing a reduction in the cytoplasmic pool of Arm, results in a similar phenotype as overexpressing full length Shotgun. This finding lends support to the notion that dissociation of the cadherin-catenin complex (CCC) may not occur at the interface between Shotgun and Arm or Arm and alpha-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 Shotgun molecules interconnecting cells, but also the stability with which they are coupled to the cytoskeleton. Biochemical studies in vertebrates and Drosophila also show that phosphorylation of the CCC does not result in increased dissociation of Arm or alpha-catenin from the CCC, suggesting that the dissociation occurs distal to alpha-catenin (Dumstrei, 2002).
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. 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. 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. However, genetic studies have 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 Shotgun, reduces the cytoplasmic pool resulting in a wg minus phenotype. However, Wnt/Wg signaling seems to have no effect on the amount of membrane bound ß-catenin. Thus, in Drosophila, it appears that Shotgun 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 (Dumstrei, 2002).
The findings suggest that another signaling pathway, the Egfr pathway, is involved in modulating cadherin-mediated adhesion and thereby controls morphogenesis. 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 (Dumstrei, 2002).
In a small subset of larval eye precursors (the 'Bolwig's organ founders') 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). 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 mutants survive. Both optic lobe and Bolwig's organ express several of their normal differentiation markers, but show a characteristic 'hyperadhesive phenotype', consisting in the failure of optic Iobe invagination and Bolwig's organ separation. Based on the similarity of this phenotype to the one resulting from Shotgun overexpression, and the genetic interaction between Egfr and Shotgun mutants in the ventral ectoderm, it is proposed that Egfr activation is required in normal development to phosphorylate the CCC and thereby allows optic lobe invagination and Bolwig's organ separation to occur. This would be in line with experimental results obtained in vertebrate cell culture studies, which have 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 affects adhesion by dissociating alpha-catenin from the CCC (Dumstrei, 2002).
Co-IP data indicates that Egfr is linked to the CCC in Drosophila as well. This implies that the effect of Egfr on Shotgun 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 vertebrate 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. Phenotypically, this results in increased invasiveness of tumor cell lines, neuronal and growth cone motility. 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. Egfr also phosphorylates the CCC and forms an integral part of this complex. 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 (Dumstrei, 2002).
The tumor suppressor APC and its homologs, first identified for a role in colon cancer, negatively regulate Wnt signaling in
both oncogenesis and normal development, and play Wnt-independent roles in cytoskeletal regulation. Both Drosophila and
mammals have two APC family members. The functions of the Drosophila APCs is further explored using the larval brain
as a model. Both proteins are expressed in the brain. APC2 has a highly dynamic, asymmetric localization through the larval neuroblast cell cycle relative to known mediators of embryonic neuroblast asymmetric divisions.
Adherens junction proteins also are asymmetrically localized in neuroblasts. In addition they accumulate with APC2 and
APC1 in nerves formed by axons of the progeny of each neuroblast-ganglion mother cell cluster. APC2 and APC1 localize
to very different places when expressed in the larval brain: APC2 localizes to the cell cortex and APC1 to centrosomes and
microtubules. Despite this, they play redundant roles in the brain; while each single mutant is normal, the zygotic double mutant has severely reduced numbers of larval neuroblasts. These experiments suggest that this does not result from misregulation of Wg signaling, and thus may involve the cytoskeletal or adhesive roles of APC proteins (Akong, 2002).
One striking feature of the asymmetric localization of
APC2 is that it is present throughout the cell cycle and is
particularly strong during interphase. During embryonic
neuroblast divisions, most asymmetric markers are localized only
during mitosis. However, less is known about their localization in larval
neuroblasts. Several asymmetric markers
in larval neuroblasts were examined, and
their localization was compared with that of APC2. In embryonic
neuroblasts, the transcription factor Prospero (Pros)
and its mRNA are GMC determinants that are asymmetrically
localized to the GMC daughter. Pros protein then becomes nuclear and helps
direct cell fate. In larval neuroblasts, a similar localization is observed. Pros is not detectable in interphase neuroblasts, when the cortical APC2 crescent is strongest. A small amount of Pros transiently localizes to an asymmetric crescent during mitosis. Pros is present at low levels in GMC nuclei and at higher levels in the nuclei of ganglion cells (Akong, 2002).
Mira is basally localized in embryonic neuroblasts,
and required there for localization of Pros protein
and mRNA. In central brain neuroblasts, Mira is diffusely cytoplasmic
during interphase, when the APC2 crescent is the
strongest. As cells enter mitosis, Mira first becomes cortical and then begins to accumulate asymmetrically on
the side of the neuroblast where the daughter will be born. By metaphase, Mira asymmetry is very pronounced. The center of the Mira crescent is always precisely aligned with one spindle pole. As a result, in cells with the
spindle pointing toward the center of the APC2 crescent,
the Mira and APC2 crescents substantially overlap, while in cells in which the spindle points to the edge of the APC2 crescent, the two crescents are
offset. Mira is partitioned into the GMC during anaphase, while APC2 relocalizes to the cleavage furrow. Mira could still be detected in some GMCs, which are thought to be those that were recently born (Akong, 2002).
In contrast to Mira and Pros, Inscuteable (Insc) and
Bazooka (Baz) localize to the apical sides of embryonic
neuroblasts, where they play essential roles in asymmetric
divisions. Insc is asymmetrically localized in larval neuroblasts. Insc localizes to the side of the neuroblast opposite that of APC2 through much, if not all, of the cell cycle. Interestingly, there is a weak Insc crescent during interphase, that becomes stronger through prophase
and metaphase. During anaphase, Insc
localizes to the neuroblast cortex but not the GMC daughter. Baz localization was similar to that of Insc,
though no cortical localization during interphase was detected. During prophase and metaphase, Baz
localizes to a crescent opposite APC2, and as
the chromosomes begin to separate, Baz localizes to a tight
cap opposite the future GMC. Together, these data
confirm that larval and embryonic neuroblasts asymmetrically
localize many of the same proteins, and that APC2
localizes on the GMC side (basal) of the neuroblast, overlapping
Mira and opposite Baz and Insc, which localize apically (Akong, 2002).
Arm also localizes asymmetrically in neuroblasts. Extending this, an examination was made of the localization of Arm's adherens junction partners DE-cadherin and ß-catenin. When central brain neuroblasts undergo a sequential
series of asymmetric divisions, the GMCs remain
associated with their neuroblast mother, resulting in a cap
of GMCs in association with each neuroblast. APC2 localizes
strongly to the boundary between the neuroblast and
each GMC, and more weakly to the borders between the GMCs. APC2 is present at lower levels in ganglion cells and differentiating neurons (Akong, 2002).
The adherens junction proteins DE-cadherin, Arm, and
ß-catenin all show a striking and asymmetric localization
pattern in central brain neuroblasts. All
precisely colocalize both at the boundary between neuroblasts
and GMCs and at the boundaries between GMCs. DE-cadherin, Arm, and
ß-catenin are also all expressed in epithelial cells of the
outer proliferation center. The localization of DE-cadherin and the catenins is consistent with the idea that cadherin-catenin-based adhesion
could help ensure that GMCs remain associated with
each other, via association with their neuroblast mother (Akong, 2002).
To further explore this, how successive
GMCs are positioned relative to their older GMC sisters was examined
using two different approaches. First Mira was used to mark the newborn GMCs and DE-cadherin was used to mark the neuroblast and all of her GMC
daughters. Mira localizes to a crescent on the side of the
neuroblast where the daughter will be born (basal side), and then is
segregated into the daughter. Mira persists for some time in newborn GMCs, and it remains detectable in the other GMCs as well, thus
allowing the position of newborn GMCs to be examined relative to their older sisters. In many cases, new GMCs are clearly born at the edge of the cluster of older GMCs. This is particularly striking in neuroblasts with many progeny. It is worth noting that the cluster of daughters is three-dimensional, comprising a 'cap' of daughters in three dimensions rather than a two-dimensional line of daughters. It is thus suspected that new daughters are born near the edge of this cap (Akong, 2002).
These data suggest that neuroblasts and their GMC
progeny remain closely associated. The GMCs then divide
to form ganglion cells and ultimately neurons. The data
further suggest that these latter cells may also remain
associated and send their axons together toward targets in
the central brain. When sections were made more deeply into the
brain, below each cluster of neuroblasts and GMCs,
structures that appear to be axons were detected projecting from these
groups of cells. These axons label with Arm, DE-cadherin, and APC1. Arm also localizes to the axons of the neuropil, while DE-cadherin and APC2 are present at low levels or are absent from this structure (Akong, 2002).
Drosophila imaginal discs are monolayered epithelial invaginations that grow during larval stages and evert at metamorphosis to assemble the adult exoskeleton. They consist of columnar cells, forming the imaginal epithelium, as well as squamous cells, which constitute the peripodial epithelium and stalk (PS). A new morphogenetic/cellular mechanism for disc eversion has been uncovered. Imaginal discs evert by apposing their peripodial side to the larval epidermis and through the invasion of the larval epidermis by PS cells, which undergo a pseudo-epithelial-mesenchymal transition (PEMT). As a consequence, the PS/larval bilayer is perforated and the imaginal epithelia protrude, a process reminiscent of other developmental events, such as epithelial perforation in chordates. When eversion is completed, PS cells localize to the leading front, heading disc expansion. The JNK pathway is necessary for PS/larval cells apposition, the PEMT, and the motile activity of leading front cells (Pastor-Pareja, 2004).
One hallmark of epithelial cells is their distinct apico-basal cell polarity. This polarity depends on a set of intercellular connections, which encircle epithelial cells at the border of the apical and basal-lateral membrane domains. The cells in insect epithelial tissues are interconnected by zonula adherens (ZAs), which function in both cellular adhesion and signaling. DE-cadherin is the major constituent of the ZAs in a complex with Armadillo (Arm, ß-catenin) and Dalpha-catenin. In addition, epithelia of flies and other invertebrates exhibit septate junctions, which are located basally to the ZAs. Septate junctions prevent diffusion through the pericellular space and are functionally equivalent to vertebrate tight junctions (Pastor-Pareja, 2004).
All imaginal disc cells at the third instar larval stage presented ZAs in an apical belt. During disc eversion, however, it was found that ZAs components delocalize from the free edges of the PS cells, remaining cytoplasmic at the edges of the perforations arising through the PS/larval bilayer and in those PS cells leading the spreading of the discs over the larval tissues. As a consequence, ZAs are lost in these cells. Moreover, septate junction components, such as Coracle and Disc Large are also found to be missing from the membranes of leading front cells (Pastor-Pareja, 2004).
The loss of apico/basal polarity and adhesion of the PS cells during disc eversion is reminiscent of an epithelial-mesenchymal transition (EMT), as described for mesoderm and neural crest cells in vertebrates, and for the acquisition of the invasive phenotype in carcinomas (Pastor-Pareja, 2004).
In summary, the evagination of imaginal disc can be divided into the following sequential steps: (1) an overall positional change of the imaginal discs leading to the confrontation and apposition of the PS and the larval epidermis; (2) a regulated modulation (PEMT) of PS cells, which involves the downregulation of their cell-cell adhesion systems and allows them to move into their local neighborhood and invade the larval epithelium; (3) the fenestration of the peripodial/larval bilayer and the formation of an unbound peripodial leading front, which will direct imaginal spreading by planar cell intercalation, and (4) a bulging of the imaginal tissue (Pastor-Pareja, 2004).
Once the hole is opened, the planar intercalation of PS cells ensures that, first in the hole and later in the leading front, all four dorsal, ventral, anterior, and posterior compartments of the wing disc are represented. This mechanism also guarantees the maintenance of a continuous epithelial barrier (Pastor-Pareja, 2004).
Armadillo, the Drosophila homolog of β-catenin, plays a crucial role in both the Wingless signal transduction pathway and cadherin-mediated cell-cell adhesion, raising the possibility that Wg signaling affects cell adhesion. This study used a tissue culture system that allows conditional activation of the Wingless signaling pathway and modulation of E-cadherin expression levels. Activation of the Wingless signaling pathway leads to the accumulation of hypophosphorylated Armadillo in the cytoplasm and in cellular processes, and to a concomitant reduction of membrane-associated Armadillo. Activation of the Wingless pathway causes a loss of E-cadherin from the cell surface, reduced cell adhesion and increased spreading of the cells on the substratum. After the initial loss of E-cadherin from the cell surface, E-cadherin gene expression is increased by Wingless. It is suggested that Wingless signaling causes changes in Armadillo levels and subcellular localization that result in a transient reduction of cadherin-mediated cell adhesion, thus facilitating cell shape changes, division and movement of cells in epithelial tissues (Wodarz, 2006; full text of article).
Ommatidial rotation is a cell motility read-out of planar cell polarity (PCP) signaling in the Drosophila eye. Although the signaling aspects of PCP establishment are beginning to be unraveled, the mechanistic aspects of the associated ommatidial rotation process remain unknown. This study demonstrates that the Drosophila DE- and DN-cadherins have opposing effects on rotation. DE-cadherin promotes rotation; DE-cad mutant ommatidia rotate less than wild type or not at all. By contrast, the two DN-cadherins act to restrict this movement, with ommatidia rotating too fast in the mutants. The opposing effects of DE- and DN-cadherins result in a coordinated cellular movement, enabling ommatidia of the same stage to rotate simultaneously. Genetic interactions, phenotypic analysis and localization studies indicate that EGF-receptor and Frizzled-PCP signaling feed into the regulation of cadherin activity and localization in this context. Thus, DE- and DN-cadherins integrate inputs from at least two signaling pathways, resulting in a coordinated cell movement (Mirkovic, 2006).
Although the role for DE-cad in tissues undergoing rearrangements during
development is established, a direct role for DE-cad in cell and tissue movement has been more difficult to study in vivo owing to its essential role in
maintenance of epithelial integrity. Analysis of adult eye phenotypes of a
homozygous viable shg/DE-cad allele and a dominant-negative DE-cad
construct (DE-cadDN), expressed in the R3/R4 and later R1/R6, R7
precursors, indicate that DE-cad is required throughout the rotation process.
The ability of ommatidia to complete the precise 90° rotation directly
depends on DE-cad activity. Both the extracellular domain, responsible for
cell-cell adhesion, and the intracellular domain, linking DE-cad to the actin
cytoskeleton, are required for rotation. DE-cad associates with the actin
cytoskeleton primarily through interactions with Arm/ß-catenin. Although
ß-catenin has a dual role in cell adhesion and Wg signaling (which can be
separated), these data indicate that during ommatidial rotation
ß-catenin acts through its role in cell adhesion (Mirkovic, 2006).
Ommatidial rotation represents the final step in establishing PCP during
eye development. The direction of rotation depends on proper R3/R4 cell fate
specification, which is determined by PCP signaling. The Egfr pathway and
input by rotation-specific genes, e.g. nemo, are thought to function
in parallel to Fz-PCP signaling. An enhancement of the
sev>DE-cadDN rotation defects was observed by dose reduction in core regulatory PCP genes dgo and stbm; ommatidial under-rotation and the number of ommatidia that did not initiate rotation in
sev>DE-cadDN/dgo-/+, stbm-/+ was
comparable with the enhancement of sev>DE-cadDN by heterozygosity
of a shg null allele). The localization of PCP protein complexes at
the level of adherens junctions is consistent with the idea that PCP factors can influence DE-cad function. The mechanism of this regulation remains unclear. The
RhoA-RNAi transgene, which was expressed only in R3/R4 precursors
during the initiation of ommatidial rotation, enhanced
sev>DE-cadDN associated under-rotation defects. Although a RhoA
requirement in multiple cellular processes makes it difficult to dissect its
specific role in rotation, the specificity of the phenotype (enhanced
under-rotation in sev>DEcad/RhoAIR) suggests a role for
RhoA in the regulation of cadherin-mediated cell movement (Mirkovic, 2006).
Although Egfr signaling appears to be required for the precise 90°
rotation, its role in the process - promoting motility or antagonizing
it - has remained unclear. The genetic data suggest that Egfr signaling acts
positively to promote rotation, since a reduction in Egfr signaling enhances the
sev>DE-cadDN under-rotation phenotype. This may reflect a
positive role for Egfr signaling in the regulation of DE-cad activity or
turnover at the membrane, as suggested from human tumor cell lines. Affecting
the function of endocytic pathway components can also have an effect on
ommatidial rotation. This might be mediated by Egfr signaling, as is thought to
be the case in human cancer cells, leading to recycling and redistribution of
E-cad at the plasma membrane (Mirkovic, 2006).
Drosophila DN-cadherins, which are encoded by the adjacent
cadN and cadN2 genes, are the main cadherins expressed in
the nervous system. In developing photoreceptors they participate in axon
guidance, and in pupal eye discs they mediate terminal patterning of the
retina [through specific expression in cone cells. During
PCP establishment, DN-cad1 is concentrated at the border between R3/R4
precursors, in a pattern largely complementary to DE-cad. This suggested a
possible combinatorial role for DE-cad and DN-cad in rotation, with DN-cad
either providing a structural role in rotating clusters, or participating in
signaling cascades that regulate cell movement. Analysis of DN-cad
mutant clones in discs during rotation demonstrated a specific function;
many mutant clusters have completed rotation well before wild-type clusters of
the same stage. These data indicate that DN-cadherins function to
slow down rotation, serving an opposing function to DE-cad (Mirkovic, 2006).
The balance and complementary distribution of DE-cad and DN-cad appear
crucial for correct rotation to occur. Mild overexpression of DN-cad1 in R3/R4
(sev>DN-cad) is sufficient to interfere with the process,
possibly by affecting DE-cad levels. Consistently, DN-cad1 overexpression
enhances sev>DE-cadDN induced under-rotation and overexpression
clones of DN-cad1 cause a decrease in endogenous DE-cad levels. Alternatively, the negative effect of DN-cad on DE-cad might be through competition for
ß-catenin, since sev>DE-cadDN is partially rescued by
UAS-ArmS2, although since
sev>DN-cad is not enhanced by arm dose reduction this
appears less likely. Interestingly, sev>DN-cad is enhanced by
co-expression of full-length DE-cad and full-length Arm. These phenotypes
resemble those of a strong sev>DN-cad line, suggesting
that DN-cad is stabilized by increased levels of available Arm, and also that
co-overexpression of two cadherins may interfere with optimal turnover rate at
the membrane (Mirkovic, 2006).
In a Drosophila follicle the oocyte always occupies a posterior position among a group of sixteen germline cells. Although the importance of this cell
arrangement for the subsequent formation of the anterior-posterior axis of the embryo is well documented, the molecular mechanism responsible for the
posterior localization of the oocyte has been unknown. The homophilic adhesion molecule Shotgun has now been shown to mediate oocyte positioning. During
follicle biogenesis, Shotgun is expressed in germline (including oocyte) and surrounding follicle cells, with the highest concentration of Shotgun being
found at the interface between oocyte and posterior follicle cells. Mosaic analysis shows that Shotgun is required in both germline and follicle cells for
correct oocyte localization, indicating that germline-soma interactions may be involved in this process. By analysing the behaviour of the oocyte in follicles
with a chimaeric follicular epithelium, the position of the oocyte is seen to be determined by the position of Shotgun-expressing follicle cells, to which the
oocyte attaches itself selectively. Among the Shotgun positive follicle cells, the oocyte preferentially contacts those cells that express higher levels of
Shotgun. On the basis of these data, it is proposed that in wild-type follicles the oocyte competes successfully with its sister germline cells for contact to the
posterior follicle cells, a sorting process driven by different concentrations of Shotgun. This is the first in vivo example of a cell-sorting
process that depends on differential adhesion mediated by a cadherin (Godt, 1998).
The Drosophila gene taiman encodes a steroid hormone receptor coactivator related to AIB1. Mutations in tai cause defects in the migration of specific follicle cells, the border cells, in the Drosophila ovary. Drosophila E-cadherin (Shotgun) is required for border cell migration. To determine whether the tai migration defect might be due to reduction in Shotgun expression, egg chambers containing tai mutant clones were stained with antibodies against Shotgun. In all wild-type stages examined, Shotgun accumulates in the central, nonmigratory polar cells, as well as in the junctions between individual border cells. Shotgun colocalizes with cortical F-actin in these locations. Prior to migration, when the border cells are still part of the follicular epithelium, Shotgun also accumulates at the junctions between border cells and nurse cells. However, once the border cells leave the follicular epithelium and invade the neighboring germline cell cluster, much less Shotgun staining is evident at the junctions between the nurse cells and border cells, relative to the level between border cells or in the polar cells. When migration is complete, Shotgun accumulates again in the junctions between the border cells and the oocyte (Bai, 2000).
In tai mutant clusters, Shotgun staining is abnormally elevated at the border cell/nurse cell junctions. In contrast, in slbo mutants, Shotgun expression fails to rise at the time of migration and Shotgun immunoreactivity is only detected at high levels within the polar cells. Armadillo (Arm) colocalizes with Shotgun in wild-type and mutant border cells. The abnormal accumulation of Shotgun and Arm in tai mutants does not appear to result from increased transcription of Shotgun because overexpression of Shotgun in border cells causes neither a migration defect nor specific accumulation of cadherin staining at the border cell/nurse cell junctions. Nor does the abnormal accumulation of Shotgun and Arm appear to be simply a consequence of the migration failure. In addition to slbo, Shotgun and Arm expression were examined in border cells that fail to migrate due to mutations in the jing locus: no defect in either expression or localization of adhesion complexes was observed. Nor are defects in either Shotgun or Arm expression or localization found in border cells that fail to migrate due to expression of dominant-negative Rac (Bai, 2000).
The accumulation of Shotgun at the border cell/nurse cell boundary suggests that the role of tai in border cell migration might be to stimulate turnover of adhesion complexes during migration in order to allow forward movement. One protein believed to play a role in turnover of adhesion complexes is Focal adhesion kinase. Drosophila FAK (Fak56D) is highly enriched in the border cells during their migration, but not in the polar cells (Bai, 2000).
To determine whether Fak56D expression or localization is affected by mutations that disrupt border cell migration, wild-type and slbo mutant egg chambers were stained and the staining was compared to that of egg chambers containing tai mosaic clones. Fak56D expression is significantly reduced in slbo mutant border cells. Furthermore, the level of reduction correlates with the degree of inhibition of migration. That is, in some slbo egg chambers, border cell migration fails completely and the cells remain at the anterior tip. In such egg chambers, Fak56D expression is undetectable. In a minority of slbo mutant chambers, the cells migrate a little. In these egg chambers, Fak56D expression is reduced compared to wild type, but is detectable. In tai mutant border cells, Fak56D expression is present; however, its distribution is altered relative to wild type. Rather than being evenly distributed throughout the cytoplasm, Fak56D appears to accumulate at the would-be leading edge of the cluster. Some border cell clusters that are mutant for tai exhibit partial migration and in these clusters, the abnormal distribution of Fak56D is only slightly affected such that little Fak56D accumulation can be detected at the most posterior position within the cluster. Thus, the severity of the migration defect in tai mutants correlates with the severity of the defect in Fak56D localization (Bai, 2000).
Follicle cell clones mutant for either Nicastrin (Ncr) or Presenilin (Psn) have a more severe phenotype than that seen in Notch or Delta mutants, indicating that both proteins must have at least one additional function in these cells that is independent of their role in Notch signaling. One aspect of this phenotype is the overaccumulation of the components of the adherens junctions, DE-Cadherin, Armadillo, and alpha-catenin, and this is probably related to the fact that both alpha-catenin and the Armadillo ortholog ß-catenin associate with Psn in mammalian cells. Although neither is required for the activity of the S3 protease or gamma-secretase, loss of Psn leads to an overaccumulation of ß-catenin in Drosophila embryos and mouse epithelial cells. The precise function of Psn in ß-catenin regulation is unknown, but the overexpressed protein in Drosophila psn mutant embryos is associated with polyubiquitin-positive cytoplasmic inclusions, suggesting that Psn is required in some way to regulate Armadillo degradation. Psn also regulates the turnover of DE-Cadherin and alpha-catenin. Furthermore, Nct is necessary for this function, suggesting that it requires the formation of the high molecular weight protease complex. Since Psn is thought to mediate the proteolysis of membrane proteins, one possibility is that Psn is recruited to DE-Cadherin by binding to the catenins, and cleaves DE-Cadherin to trigger degradation. Alternatively, Psn could regulate the turnover of the catenins in some other way, and their overaccumulation in psn and nct mutants might then lead to the stabilization of Cadherin complexes at the membrane (López-Schier, 2002).
Drosophila hematopoiesis occurs in a specialized organ called the lymph gland.
In this systematic analysis of lymph gland structure and gene expression, the developmental steps in the maturation of blood cells (hemocytes) from
their precursors are defined. In particular, distinct zones of hemocyte maturation,
signaling and proliferation in the lymph gland during hematopoietic progression
are described. Different stages of hemocyte development have been classified
according to marker expression and placed within developmental niches: a
medullary zone for quiescent prohemocytes, a cortical zone for maturing
hemocytes and a zone called the posterior signaling center for specialized
signaling hemocytes. This establishes a framework for the identification of
Drosophila blood cells, at various stages of maturation, and provides a genetic
basis for spatial and temporal events that govern hemocyte development. The
cellular events identified in this analysis further establish Drosophila as a
model system for hematopoiesis (Jung, 2005).
In the late embryo, the lymph gland consists of a single pair of lobes
containing ~20 cells each. These express the transcription factors Srp and
Odd skipped (Odd),
and each cluster of hemocyte precursors is followed by a string of
Odd-expressing pericardial cells that are proposed to have nephrocyte
function. These lymph gland lobes are arranged bilaterally such that they
flank the dorsal vessel, the simple aorta/heart tube of the open circulatory
system, at the midline. By the second larval instar, lymph gland morphology is
distinctly different in that two or three new pairs of posterior lobes have
formed and the primary lobes have increased in size approximately tenfold (to
~200 cells. By the late third instar, the lymph gland has grown significantly in size
(approximately another tenfold) but the arrangement of the lobes and
pericardial cells has remained the same. The cells of the third instar lymph
gland continue to express Srp (Jung, 2005).
The third instar lymph gland also exhibits a strong, branching network of
extracellular matrix (ECM) throughout the primary lobe. This network was
visualized using several GFP-trap lines in which GFP is fused to endogenous
proteins. For
example, line G454 represents an insertion into the viking
locus, which encodes a Collagen IV component of the extracellular matrix.
The hemocytes in the primary lobes of G454
(expressing Viking-GFP) appear to be clustered into small populations within
pockets or chambers bounded by GFP-labeled branches of various sizes. Other lines, such as
the uncharacterized GFP-trap line ZCL2867, also highlight this
branching pattern. What role this intricate ECM network plays in
hematopoiesis, as well as why multiple cells cluster within these ECM
chambers, remains to be determined (Jung, 2005).
Careful examination of dissected, late third-instar lymph glands by
differential interference contrast (DIC) microscopy revealed the presence of
two structurally distinct regions within the primary lymph gland lobes that
have not been previously described. The periphery of the primary lobe generally exhibits a
granular appearance, whereas the medial region looks smooth and compact. These
characteristics were examined further with confocal microscopy using a
GFP-trap line G147, in which GFP is fused to a microtubule-associated
protein. The G147 line is expressed throughout the lymph gland but, in
contrast to nuclear markers such as Srp and Odd, distinguishes morphological
differences among cells because the GFP-fusion protein is expressed in the
cytoplasm in association with the microtubule network. Cells in the
periphery of the lymph gland make relatively few cell-cell contacts, thereby
giving rise to gaps and voids among the cells within this region. This
cellular individu