armadillo


DEVELOPMENTAL BIOLOGY

Embryonic

ARM protein is uniformly distributed at the cellular blastoderm stage. In early gastrulation, ARM accumulates at high levels in the procephalic lobe and the posterior midgut. At early germ band extension the first ARM stripes appear along the ventral side of the most anterior segments. At mid-germ band extension, ARM is present in 15 stripes in parasegments 0-14. High levels are apparent in the prospective labral and antennal regions, and the stomodeum and proctodeum (Riggleman, 1990). In the nervous system of Drosophila embryos, ARM and Actin are enriched in fiber tracts (Peifer, 1990).

Drosophila Armadillo and its vertebrate homolog beta-catenin have dual roles in epithelial cells: transducing signals from the Wingless/Wnt family of proteins and working with cadherins to mediate cell adhesion. Wingless/Wnt signaling also directs certain cell fates in the central nervous system (CNS), and cadherins and catenins are thought to function together during neural development. An analysis of the biochemical properties of a second Armadillo isoform, with a truncated carboxyl terminus generated by alternative splicing, was carried out. This isoform accumulates in differentiating neurons. Using armadillo alleles that selectively inactivate the cell adhesion or the Wingless signaling functions of Armadillo, Armadillo was found to have two sequential roles in neural development. Armadillo function in Wingless signal transduction is required early in development for determination of neuroblast fate. Later in development, disruption of the cell-cell adhesion function of Armadillo results in subtle defects in the construction of the axonal scaffold. Mutations in the gene encoding the Drosophila tyrosine kinase Abelson substantially enhance the severity of the CNS phenotype of armadillo mutations, consistent with these proteins functioning co-operatively at adherens junctions in both the CNS and the epidermis. This is one of the first demonstrations of a role for the cadherin-catenin system in the normal development of the CNS. The genetic interactions between armadillo and abelson point to a possible role for the tyrosine kinase Abelson in cell-cell adhesive junctions in both the CNS and the epidermis (Loureiro, 1998).

Nullo, Armadillo, cellularization and junctions

During cellularization, the Drosophila embryo undergoes a large-scale cytokinetic event that packages thousands of syncytial nuclei into individual cells, resulting in the de novo formation of an epithelial monolayer in the cortex of the embryo. The formation of adherens junctions is one of the many aspects of epithelial polarity that is established during cellularization: at the onset of cellularization, the Drosophila ß-catenin homolog Armadillo (Arm) accumulates at the leading edge of the cleavage furrow, and later to the apicolateral region where the zonula adherens precursors are formed. The basal accumulation of Arm colocalizes with E-cadherin and alpha-catenin, and corresponds to a region of tight membrane association, which is referred to as the basal junction. Although the two junctions (apicolateral and basal) are similar in components and function, they differ in their response to the novel cellularization protein Nullo. The nullo gene encodes a predicted protein of 213 amino acids, a large proportion of which is basic (Rose, 1992). nullo transcripts are first detectable at nuclear cell cycle 11, peak in accumulation at the end of cycle 13, and disappear rapidly as cellularization begins. Nullo is present in the basal junction and is required for its formation at the onset of cellularization. In contrast, Nullo is degraded before apical junction formation, and prolonged expression of Nullo blocks the apical clustering of junctional components, leading to morphological defects in the developing embryo. These observations reveal differences in the formation of the apical and basal junctions, and offer insight into the role of Nullo in basal junction formation (Hunter, 2000).

During cellularization, the syncytial blastoderm is converted to a monolayer of cells that display many of the features of a polarized epithelium, including distinct apical and basolateral surfaces that are separated by a belt-like ZA. The formation of the ZA has been well documented: it begins with an accumulation of Arm and E-cadherin to spot-like adhesive contacts in the newly formed apicolateral membrane. During late cellularization, these accumulations cluster apically to form the apical spot-junctions, which give rise to the ZA during gastrulation. In addition to its apical accumulation at the ZA, Arm protein is also localized to the leading edge of the cleavage furrows. This localization is observed at the onset of cellularization and is maintained as the membrane invaginates into the interior. When cellularization is completed, this early Arm accumulation is found at the basal-most region of the lateral membrane. It was of interest to enquire whether the localization of Arm to the basal tip of the cleavage furrow might indicate a novel requirement for adherens junctions during cellularization. As in a traditional adherens junction, the Arm protein at the cellularization front colocalizes with E-cadherin and alpha-catenin, and electron micrographs show that the membranes just above the furrow canal are more closely apposed than other regions of the lateral membranes. Although this region lacks the electron dense plaques of a mature ZA, it is similar in nature to the cell-cell contacts seen at the apical spot-junctions; this adhesive zone is therefore referred to as the basal junction (Hunter, 2000).

To position the basal junction relative to the furrow canal, the localization of Arm with respect to myosin in the cleavage furrow was examined. In cross section, embryos initiating cellularization show spot-like accumulations of Arm at the sites of somatic bud contact. This is similar to the accumulation of Arm seen in pseudo-cleavage furrows. The spots of Arm staining in the nascent cleavage furrows are spatially distinct from the early accumulation of myosin, suggesting the basal junction and furrow canal form as separate domains. Many embryos that have a clear accumulation of Arm, but lack detectable levels of myosin: this suggests that the formation of the basal junction might precede the completion of myosin localization to the cellularization front. As cleavage furrows extend, it is clear that myosin and Arm are present in nonoverlapping regions of the cellularization front. During late cellularization, the furrow canals widen and it becomes apparent that the basal Arm population marks the boundary between the existing lateral membrane and the expanding basal membrane. At this stage, embryos also begin to accumulate Arm at the apical junction. As the embryo initiates gastrulation, the basal accumulation of Arm, and other junctional proteins, is gradually lost, while the apical population continues to coalesce into the mature ZA (Hunter, 2000).

In summary, cleavage furrows of the Drosophila blastoderm form two distinct adherens junctions: a transient junction at the boundary of the basal and lateral membrane domains, and a permanent junction at the boundary of the presumptive apical and basolateral domains. Like the apical-spot junction, the basal junction contains coincident accumulations of Arm, E-cadherin, and alpha-catenin, and corresponds to a region of tight membrane apposition (Hunter, 2000).

The rapid rate of cellularization requires that the apical and basal junctions be formed in close spatial and temporal proximity, without coalescing to form a single junctional complex. The first indication of how this might be achieved came from observations of Arm protein distribution in nullo mutant embryos. In nullo mutant embryos, a subset of somatic bud contacts fails to accumulate actin and myosin, and no cleavage furrows form at these positions (Simpson, 1990). The distribution of myosin and Arm in the remaining furrows was examined, and it was found that myosin shows its typical localization to the furrow canal, but the early Arm protein is not restricted to the basal junction. Instead, Arm extends apically along the lateral membrane, suggesting that in the absence of Nullo protein, the basal Arm population moves toward the apical junction. Interestingly, the apical junction is not affected by the nullo mutation: during late cellularization the existing cleavage furrows form apical spot-junctions that coalesce to form ZAs. The observation that Nullo is required for basal, but not apical, junction formation is supported by the fact that Nullo protein is normally found at the basal tip of the cleavage furrow, and is degraded before apical junction formation (Hunter, 2000).

To pinpoint the onset of the basal junction defects, the distribution of Arm protein was examined in embryos initiating cellularization. Since wild-type and nullo mutant embryos are phenotypically identical at this stage, anti-HA-Nullo immunostaining was used to identify the embryos. During the first phase of cellularization, cleavage furrows form at the slight infoldings of membrane where adjacent somatic buds abut each other. Surface views of wild-type embryos during this stage show a diffuse hexagonal pattern of Arm that corresponds to the infoldings of plasma membrane. The Arm staining gradually resolves into sharp lines as basal junctions form, but this does not occur synchronously across the embryo. At early stages a given region contains both diffuse and sharp lines of Arm staining. This process is completed by the onset of cleavage furrow invagination, at which point all of the cleavage furrows have sharp lines of Arm accumulation at the level of the basal junction. As in apical junctions, this staining is strongest at the lateral cell-cell contacts, and Arm is depleted from the vertices of the hexagonal array (Hunter, 2000).

nullo mutant embryos show a normal formation of somatic buds above each nucleus and the same diffuse pattern of Arm protein as wild-type embryos. Arm also begins the same gradual transition to form sharp lines of staining at the level of the basal junction. However, some interfaces fail to establish a focused concentration of Arm protein, so that the partially resolved Arm network characteristic of early stages is still present when the cleavage furrows begin to invaginate. Those regions that fail to form a basal junction do not give rise to cleavage furrows. Areas with a basal junction invaginate, but Arm protein does not remain restricted to the basal junction, as described above. It is interesting to note that the defects in Arm distribution can be observed before nullo mutant embryos develop visible morphological defects, and often can be seen before the visible accumulation of myosin to the furrow canals. This may suggest that the failure to establish a basal junction is the primary defect in nullo mutant embryos (Hunter, 2000).

Based on these observations, it is proposed that the Nullo protein is required to maintain the early accumulation of Arm at the basal junction. In the absence of Nullo, a subset of somatic bud contacts contains only a diffuse accumulation of Arm protein, and fails to initiate cleavage furrows. In those furrows that do invaginate, Arm protein is not restricted to the basal junction but spreads apically along the nascent lateral membrane (Hunter, 2000).

The nullo mutation was originally thought to primarily affect the actin-myosin network that forms at the cellularization front. Given the apparent involvement of nullo in the formation of the basal junction, the localization of Nullo with respect to actin, myosin, and Arm was examined. The Nullo protein had been shown to be concentrated at the leading edge of the cleavage furrow during cellularization (Postner, 1994), but the fixation conditions required to detect the protein precluded most colocalization studies. Therefore a nullo transgene was constructed containing a triple-HA tag at the COOH terminus. The protein produced by this transgene rescues the nullo mutant phenotype and shows the same temporal and spatial localization as the wild-type protein, allowing Nullo to be detected under a wider range of conditions (Hunter, 2000).

A comparison of Nullo and actin shows a strong colocalization during early cellularization. Both Nullo and actin are initially distributed apically, beneath the surface of the somatic buds, and then localize to the nascent cleavage furrow as it begins to invaginate. Nullo and actin maintain their colocalization at the cellularization front until Nullo is degraded during late cellularization. To determine if the concentration of Nullo at the cellularization front corresponds to the basal junction, the furrow canal, or both, the distribution of Nullo was examined with respect to myosin and Arm. It was found that Nullo and Arm protein distributions overlap at the basal junction, whereas the region just below this contains only Nullo protein. Counterstaining with myosin confirms that this region corresponds to a population of Nullo protein in the furrow canal. Thus, the Nullo protein colocalizes not only with actin and myosin in the furrow canal, but also with actin and Arm at the basal junction (Hunter, 2000).

The colocalization of Nullo with actin, Arm, and myosin is maintained until late cellularization, at which point Nullo protein is rapidly degraded. At the onset of gastrulation, the basal junction is also lost, and myosin relocalizes from the furrow canal to the apical region of the cell. This first occurs in the cells that form the ventral furrow and therefore it was of interest to see whether Nullo protein is also degraded more rapidly in these cells. By examining embryos in cross-section, the loss of Nullo protein from the cellularization front was observed to occur more rapidly on the ventral surface of the embryo (Hunter, 2000).

Ectopic Nullo expression gives rise to a striking defect in the later localization of Arm to the apical junctions. The first differences are observed at the point when wild-type embryos lose Nullo protein and accumulate Arm along the apicolateral surface. At this stage, embryos expressing UASnullo maintain a normal localization of Arm at the basal junction, but fail to establish a concentrated localization of Arm in the apicolateral region. Instead, low levels of Arm protein are distributed along a broad region of the lateral membrane, and fail to coalesce into a junctional structure during gastrulatio. Similar defects are also observed in the distribution of alpha-catenin and E-cadherin (Hunter, 2000).

The junctional defects lead to irregularities in cell morphology and a failure to form the ventral furrow. Although the ventral cells of embryos expressing ectopic Nullo undergo a normal basal to apical shift in myosin localization and rapidly lose the basal accumulation of Arm, they are unable to invaginate. The cells do appear to initiate cell-shape changes and occasionally produce a wide, shallow furrow on the ventral surface, but they fail to complete ventral furrow formation. In contrast, the cephalic furrow, which forms at the same time, appears normal. Since the ventral surface is normally the first region to degrade Nullo protein, it may be especially sensitive to continued Nullo expression. This may indicate that the rapid, coordinated cell constriction that forms the ventral furrow has a more stringent requirement for apical spot-junctions than other movements of early gastrulation (Hunter, 2000).

These findings suggest that the rapid degradation of Nullo protein in late cellularization is critical for the establishment of the apical junction and the formation of the ventral furrow. Although Nullo protein is required to stabilize the accumulation of Arm in the basal junction, it appears to block the coalescence of Arm that gives rise to the apical spot-junctions. It is also shown that ectopic Nullo blocks only the de novo formation of apical adherens junctions that occurs as epithelial polarity is first established during cellularization (Hunter, 2000).

The formation of an adherens junction between the basal and lateral membrane compartments is unusual, and may reflect a unique need to separate these membrane domains during cellularization. The cytokinesis that takes place during cellularization is a two-step process: the lateral membrane is generated during the initial invagination of the cleavage furrow and the basal membrane is produced by the later expansion of the furrow canal. Therefore, the basal membrane of the furrow canal must be isolated as the lateral cell surface elongates. The furrow canal is known to constitute a separate membrane domain (Lecuit, 2000) that accumulates a specific set of proteins. It also maintains a larger intercellular space, which may prevent lateral contacts that could block furrow canal expansion. In this respect, the basal junction separates the noncontacting basal membrane from the adherent lateral membrane in a manner similar to the separation of the apical (noncontacting) and lateral (contacting) membrane by the ZA. The tight adhesion at the basal junction may also insulate the nascent lateral junctions from the outward pull that generates the basal cell surface. The basal junction therefore acts to define membrane domains and reinforce cell-cell contact in a manner similar to the traditional apical adherens junction (Hunter, 2000 and references therein).

During embryonic development, spot-junctions are often created by the delivery of cadherin-catenin complexes to regions of cell-cell contact. This appears to be the case for the basal junction: unlike Nullo and actin, which are initially present along the apical surface, Arm first appears as dots of staining at sites where somatic buds abut. The lack of overlap between Arm and myosin suggests that Arm is restricted to the small region of membrane contact between the embryo surface and the noncontacting domain of the furrow canal. This small area of localization may allow junctional components to bypass the clustering step that typically follows the delivery of the cadherin-catenin complexes. Examination of the basal Arm domain reveals that its size does not change appreciably between the onset of cleavage furrow invagination and late cellularization (Hunter, 2000).

The absence of the clustering step may, in fact, be critical for the formation of the basal junctions. Unlike a typical adherens junction, which is formed at sites of cell-cell contact, the basal junction forms at sites where a single membrane folds inward and contacts itself. Junctional complexes form on opposite sides of the shallow fold and establish extracellular contacts, but they are also separated by an extremely small intracellular space. In this situation, clustering is problematic: it might allow the two sides of a junction to collapse into a single complex, or allow the recruitment of cadherins and catenins into neighboring furrow canals. A similar problem is faced in mid-cellularization, when the coalescence of the apical junction takes place in close proximity to the existing basal junction (Hunter, 2000).

The Drosophila embryo must have a mechanism to preserve the local accumulations of junctional components when multiple adherens junctions are formed within a common membrane. It is proposed that the presence of Nullo stabilizes the accumulation of Arm in the basal junctions and prevents its recruitment into neighboring complexes or the coalescing apical junctions. In the absence of Nullo a subset of furrows fails to focus Arm protein into a stable basal junction, perhaps due to the recruitment of junctional components into neighboring complexes. The remaining basal junctions elongate during cellularization, suggesting that Arm is being recruited into the coalescing apical junctions. Nullo does not appear to be required for maintenance of the basal junction once it has moved below the region of apical junction synthesis. Although Nullo is degraded during mid-cellularization, the basal junction persists into early gastrulation, and its life is not extended in the presence of prolonged Nullo expression (Hunter, 2000).

A striking feature of nullo is its stringent developmental regulation: by mid-cellularization nullo gene expression has ceased, and the Nullo protein is rapidly degraded (Simpson, 1990). Extending the period of nullo expression into late cellularization prevents the formation of the apical adherens junctions. Instead, Arm, alpha-catenin, and E-cadherin accumulate along a broad apicolateral region, which appears to correspond to the zone where new membrane is inserted into the cleavage furrow. This suggests that the junctional components are delivered to the lateral membrane, but fail to cluster towards the apicolateral boundary. It is proposed that, as in basal junction formation, the ectopic Nullo protein stabilizes the accumulation of cadherins and catenins as they are delivered to regions of lateral membrane contact. Although the depth of the contacting membrane is <1 µm when the basal junction is formed, it has expanded to >20 µm by the onset of apical junction formation. Therefore, cadherins and catenins targeted to this large area must undergo conventional clustering movements to form a concentrated accumulation at the apicolateral boundary. The continued presence of Nullo protein blocks this clustering, and instead preserves the transitional state in which junctional components are broadly distributed along the lateral membrane. Ectopic expression of Nullo during late embryogenesis does not disrupt development, suggesting that once epithelial polarity is established, the presence of Nullo does not affect adherens junctions. The existence of mature apical and basolateral domains may provide cues for the targeting of cadherins and catenins, making the formation of subsequent junctions less reliant on large clustering movements, and therefore less susceptible to the effects of Nullo protein (Hunter, 2000).

Armadillo levels are reduced during mitosis in Drosophila

The Armadillo protein of Drosophila is both a structural component of adherens junctions at apical cell membranes and also a key cytoplasmic transducer of the Wingless signalling pathway. The Gal4-UAS system has been used to over-express Armadillo in the wing: this hyperactivates the Wingless pathway and leads to the formation of ectopic, supernumerary wing bristles. This adult phenotype is dominantly enhanced by mutations in string and, conversely, is suppressed by co-expression of String. Furthermore, the steady state levels of Armadillo protein produced from the UAS transgene are also sensitive to string dosage in the cells of the larval imaginal wing disc. Consistent with the role of String in promoting mitosis and with the genetic interaction data, a strong correlation is found between progression through mitosis and a reduction in Armadillo levels. Significantly, this is true whether Armadillo is over-expressed or not, and both cytoplasmic (signalling) and membrane-associated (junctional) Armadillo appears to be affected. It is concluded that this phenomenon may reduce the efficacy of Wingless signalling and/or intercellular adhesion during cell division (Marygold, 2003).

Thus String has gene dosage-dependent effects on Wg-Arm signalling in the adult wing and on Arm protein stability in imaginal wing disc cells. The nature of these interactions suggest that Stg behaves as a negative regulator of Wg-Arm signalling: over-expression of Stg lowers Arm levels and inhibits Wg signal transduction, while a reduction of stg dosage has the opposite effect. Stg is the major mitotic inducer in Drosophila cells. Consistent with this role, it is found that inhibition of mitoses in imaginal wing discs increases the number of cells containing high Arm levels, while induction of mitoses across the disc reduces Arm levels. At the cellular level, high cytoplasmic Arm expression is only detected in cells which are in interphase, while cells undergoing mitosis show reduced levels of cytoplasmic Arm. Taken together, these data suggest that cytoplasmic Arm is destabilized or degraded significantly during Stg-mediated mitosis and that this effect can attenuate Wg signalling (Marygold, 2003).

When looking at the level of cellular resolution, it was found that the correlation between interphase/mitosis and, respectively, high/low cytoplasmic Arm levels is not absolute. (1) An interphase state does not necessarily predict high Arm expression, and conversely, low Arm levels are not found solely in mitotic cells. Clearly factors other than cell cycle phasing (such as the degree of Wg reception) influence Arm stabilization. Nonetheless, passage through mitosis results in a detectable and tangible decrease in Arm stability and Wg signalling. (2) Although high levels of cytoplasmic Arm are never observed in mitotic cells identified by their characteristic cell shape changes, rare mitotic cells were observed that had relatively high cytoplasmic Arm. This apparent discrepancy may be explained if Arm is destroyed only during a late phase of mitotic progression: mitotic cells only take on an ovoid, elongate appearance during late anaphase and telophase. These observations fit with a model in which cytoplasmic Arm is specifically degraded at or after the metaphase-anaphase transition (Marygold, 2003).

At the present time, the precise molecular mechanism whereby progression through mitosis leads to a reduction in cytoplasmic Arm levels is not understood. Nonetheless, it is reasoned that the Stg-dependent effects observed must be caused through the ability of Stg to remove inhibitory phosphate from Cdk1 and thereby induce mitotic entry. This is because: (1) Cdk1 is the only reported target of Stg; (2) the sole function ascribed to Stg is to promote G2-M progression, and (3) passage through mitosis correlates with reduced Arm levels in these assays. It is therefore concluded that the decreased stability of cytoplasmic Arm during mitosis occurs as a result of increased Cdk1 activity, though it is not known how direct this effect may be. One attractive possibility is that Arm itself is targeted for proteolysis at the metaphase-anaphase transition of mitosis via the anaphase-promoting complex/cyclosome (APC/C), although the Arm protein lacks the canonical sequence recognition motifs that are found in known APC/C substrates. Alternatively, an upstream component of the canonical Wg pathway could be specifically regulated during mitosis such that cytoplasmic Arm is destabilized as a result (Marygold, 2003).

Regardless of the exact mechanism(s) by which passage through mitosis lowers cytoplasmic Arm, it is interesting to ask why varying the gene dosage of stg should modify the sensitized phenotypes of ectopic Arm? Genetic manipulations of Stg activity might feasibly affect the relative number of Cdk1 molecules that are dephosphorylated by Stg at mitosis. Over-expression of Stg, for example, could cause hyperactivation of Cdk1, leading to excessive phosphorylation of Cdk1 substrates (such as the APC/C) and, ultimately, a near-complete destruction of the over-expressed Arm. Alternatively, as Stg is the limiting inducer of mitosis in Drosophila cell cycles, manipulating stg dosage may have a more significant effect on the absolute or relative length of time cells spend in mitosis and/or interphase. Such effects could thus impinge on the amount of over-expressed Arm that is degraded during mitosis and the degree of productive Wg signalling during interphase. In this regard, it is interesting to note that mutations in genes encoding other, non-limiting promoters of the G2-M transition (including Cdk1, Cyclin A, Cyclin B and Cyclin B3) do not interact in the En>Arm-based assays (Marygold, 2003).

Why should Arm be degraded at mitosis? Several models suggest possible answers. (1) It may be that intercellular adhesion needs to be relaxed during cell division and that junctional Arm is the primary target of destruction during mitosis, with cytoplasmic Arm being degraded only fortuitously. The typical rounding up of dividing cells in culture may indeed reflect a general decrease in Arm/ß-catenin-mediated adhesion. However, it is unclear how the usually stable junctional Arm would be destroyed in this scenario. (2) If some aspects of Wg signalling antagonized mitotic progression, then mitotic degradation of cytoplasmic Arm could be a mechanism employed by the cell to reduce such inhibition and thus allow cell division to continue on cue. Indeed, Wg signalling at the D/V boundary of the Drosophila wing disc is known to inhibit mitotic entry. However, mitotic down-regulation of Wg signalling cannot be absolutely necessary for mitosis to proceed, since cells over-expressing non-degradable forms of Arm still appear to divide and proliferate. (3) Cell cycle-intrinsic attenuation of Wg signalling/Arm levels could serve to reflect the proliferation status of a cell. Healthy and actively proliferating cells would repeatedly lower their cytoplasmic Arm levels at each and every mitosis, whereas a sick, slow-growing cell would accumulate relatively higher Arm levels during their extended interphase. If imaginal disc cells could detect the relative level of Wg transduction/Arm levels in neighboring cells, this mechanism could allow the identification and elimination of such relatively slow-growing cells within the disc, as in the process known as 'cell competition'. Clearly, the future challenge is to discover which of these possibilities is correct (Marygold, 2003).

Wnt, Hedgehog and junctional Armadillo/beta-catenin establish planar polarity in the Drosophila embryo

To generate specialized structures, cells must obtain positional and directional information. In multi-cellular organisms, cells use the non-canonical Wnt or planar cell polarity (PCP) signaling pathway to establish directionality within a cell. In vertebrates, several Wnt molecules have been proposed as permissible polarity signals, but none has been shown to provide a directional cue. While PCP signaling components are conserved from human to fly, no PCP ligands have been reported in Drosophila. This paper reports that in the epidermis of the Drosophila embryo two signaling molecules, Hedgehog (Hh) and Wingless, provide directional cues that induce the proper orientation of Actin-rich structures in the larval cuticle. Proper polarity in the late embryo also involves the asymmetric distribution and phosphorylation of Armadillo (Arm or β-catenin) at the membrane and that interference with this Arm phosphorylation leads to polarity defects. These results suggest new roles for Hh and Wg as instructive polarizing cues that help establish directionality within a cell sheet, and a new polarity-signaling role for the membrane fraction of the oncoprotein Arm (Colosimo, 2006).

These results indicate that Wg and Hh act as instructive cues in the Drosophila embryonic epidermis to establish planar cell polarity. Though the complete molecular mechanisms that control the complex system of PCP in the ventral epidermis remain to be determined, this process appears to occur in part through the asymmetric localization of Arm at the membrane. Further, proper polarity signaling is abolished if specific phosphorylation sites within the alpha-catenin binding domain of Arm are mutated. These sites were originally found to increase the affinity of β-catenin for alpha-catenin when phosphorylated by Casein Kinase II in vitro, suggesting one mechanism for stabilizing junctions. These findings provide in vivo support for this hypothesis, since low levels of ArmAA Arm in which two threonines were mutated to alanines) rescues cellular junction defects to a similar extent as expression of an alpha-catenin/E-cadherin fusion protein, a protein that makes overly stable junctions. Higher levels of ArmAA expression lead to apparent polarity defects. Since ArmAA does not localize asymmetrically the way that wild-type Arm does, it is inferred that CKII phosphorylation may be required for the accumulation of junctions in specific regions of cells implying that stable junctions at specific sites in a cell are required for proper planar cell polarity. Further, these findings revealed that when all signaling activity is abolished through null mutations in the Wg or Hh signaling pathways, both cell identity and polarity determination was disrupted. It remains to be determined how Wg and Arm proteins function in polarity signaling, specifically whether they work through known PCP components, function similarly to their role in dorsal closure, or perhaps through novel signaling mechanisms like the interaction with Notch or Axin (Colosimo, 2006).

The wg and hh genes are required for the proper establishment of cell identities within segments. Uniform expression of Wg in the embryo leads to a completely naked cuticle, but short early bursts of expression establish what appears to be relatively normal patterning. Upon closer inspection, however, the denticle orientations of these early expression rescue experiments do not entirely resemble the wild-type patterning. This suggests that early expression of Wg can rescue several aspects of cell identity, including development of naked cuticle, but Wg is also required in the later stages when denticles form to specify proper orientations. Expression of ectopic Wg has been observed to correlate with denticles pointing toward the source of Wg, and expression of ectopic Hh also leads to denticles pointing away from its source. These previous studies, however could not distinguish between cell fate transformation and changes in cell polarity since the sources of both ligands were in the normal orientation. The current observations argue that Hh and Wg can have direct effects on cell polarity since denticles and their precursors (the Actin foci) are rotated 90° away from the anterior-posterior axis corresponding to the direction of ligand expression (Colosimo, 2006).

In the early embryo, expression of Wg and Hh is determined by pair-rule genes, but this effect is transient and requires mutually reinforcing positive activation loops to form between cells expressing Wg and En/Hh. This is the early signaling event that establishes an organizer region in each parasegment. Therefore, if either Hh or Wg is missing, expression of both is lost. The early effects of Hh and Wg expression are important for the establishment of segment boundaries, and these boundaries function in limiting Wg function, giving this morphogen an asymmetric range. The current findings agree with these observations, because it was observed that the Wg effect is best observed when hh is absent, suggesting that when the hh gene is present a boundary may be formed, thus preventing Wg from orienting the denticles to the same extent. It also appears that the distance over which Wg can act is longer in the absence of hh as expected from previous observations. According to the proposed boundary model, the extent of Wg influence is to the first denticle-secreting cell, but not beyond. This finding, along with the discovery that denticles orient toward the source of Wg, may explain why the first row of denticles in wild-type larvae points toward the anterior of the embryo. Only this row of cells receives Wg signal as the segment boundary blocks further action by Wg to the next row of cells. In contrast, Hh can and does affect the next two rows of cells. It was found that expression of Hh causes a rotation away from the source, and could explain why the next two rows of denticles point toward the posterior of the embryo. These results do not explain the final orientation of all rows of denticles, and one likely complication is that in late embryonic stages the Notch and EGFR signaling pathways affect the identities of cells within the denticle belt. It will be interesting to test what effects these signals have on the final orientation of the orientation of denticles, and whether the Notch pathway functions in polarity as well (Colosimo, 2006).

The PCP signaling pathway determines planar polarity in a variety of tissues. In vertebrate and C. elegans studies, Wnts have been implicated in the establishment of polarity, but only one study in Drosophila suggested a role for Wg in PCP (Price, 2006). In fact, the present model excludes the known morphogens, and suggests that PCP is established through cell-cell interactions involving atypical cadherins like Flamingo or through an as yet unidentified factor X. Though this study does not address the function of the known components of PCP signaling in the embryo, it is interesting that mutants in PCP signaling pathway components affect the polarity of the first two rows of denticles. The current findings support the possibility that Wg and Hh lead to the expression of an unknown factor affecting the polarization of denticles, because blocking the transcriptional readout of either Wg or Hh with tcf or ci mutations respectively prevents the polarizing activity of both pathways. This is similar to the PCP disruptions found in the Drosophila eye model for Wg signaling components. The current observations do, however, offer a further possibility, namely that by blocking all Wg signaling with null mutations the underlying polarity organizing function of Wg may be obscured. In the weak armF1A mutant the orientation of denticles can be affected by the expression of Wg without affecting the cell-fates, suggesting that perhaps Wg can affect polarity directly. This effect of Wg was not observed in stronger arm mutant embryos suggesting that Arm protein is required for the Wg effect on denticle orientation. Interestingly, cell culture work has recently implicated Wg in controlling adherens junction strength (Colosimo, 2006).

The use of the embryonic epidermis led to the the interesting possibility that Arm functions in cell polarity. Since some of the molecules involved in the PCP signaling pathway are similar to Cadherins, it seems logical that adhesion is involved in the establishment of polarity. However, adherens junctions have not been implicated so far. This is likely due to the difficulty of working with adherens junction component mutations that are often cell-lethal in the systems that have been used to study PCP. Use of the embryo allows relatively simple perturbation of arm function, and efficient ubiquitous or directional ectopic expression. Unfortunately, the major limitation of the ventral midline expression assay is that it only works for secreted, diffusible ligands. Thus, cell-autonomous activation of Hh or Wg pathway components (such as with activated Arm or Smo) along the ventral midline cannot be observed, since these cells invaginate and do not become a part of the external epidermis (Colosimo, 2006).

The fact that β-catenin is both an oncogene and a component of adherens junctions has led to many studies attempting to link the phosphorylation state of β-catenin in adherens junctions to the epithelial to mesenchymal transition (EMT) in cancer cells and during development. Phosphorylation of tyrosine residues in β-catenin is thought to lead to disassembly of adherens junctions, but recent studies both in vivo and in vitro have challenged this. Certainly these discrepancies will have to be resolved, but this study provides evidence for a different mechanism for regulating junctions, and perhaps EMT, through threonine phosphorylation-based stabilization or dephosphorylation-based destabilization of junctions. It will be crucial to establish which is the regulated step, and whether there are any phosphatases involved in this process in addition to the known kinase CKII (Colosimo, 2006).

Interestingly, the recent findings that alpha-catenin and ß-catenin do not form a stable complex in junctions, suggests a possible explanation for these findings. It is speculated that expression of ArmAA can rescue the basic activity of junctions lost in strong arm mutant embryos, which is to hold a tissue together. However, its reduced affinity for alpha-catenin does not cause a local increase in alpha-catenin levels and therefore Actin levels do not become asymmetric. This leads to a skewing of the normal polarization of the Actin cytoskeleton. It will be crucial to determine how junctions are localized asymmetrically in the first place, and whether this is dependent on extracellular signaling. These findings, and the effects of alpha-catenin mutations on inflammation and tumor progression in the mouse epidermis make analysis of the interaction between alpha- and β-catenin particularly important (Colosimo, 2006).

These experiments provide some of the first evidence that the Hh signaling pathway is involved in polarity. It is particularly interesting that Hh expression leads to the reorganization of Actin structures within epithelial cells, since this suggests that Hh can affect the polarity of the Actin cytoskeleton. This finding is also relevant to cancer biology, because during metastasis, cancer cells lose polarity and essentially ignore their environment. These results show that Wnts and Hh can affect cell polarity, in addition to their well-known effects on cell proliferation. Along with the recent report that TGFβ signaling affects polarity and EMT, these findings imply that this dual role may be a general feature of oncogenic signaling pathways (Colosimo, 2006).

Components of Intraflagellar Transport complex A function independently of the cilium to regulate canonical Wnt signaling in Drosophila.

The development of multicellular organisms requires the precisely coordinated regulation of an evolutionarily conserved group of signaling pathways. Temporal and spatial control of these signaling cascades is achieved through networks of regulatory proteins, segregation of pathway components in specific subcellular compartments, or both. In vertebrates, dysregulation of primary cilia function has been strongly linked to developmental signaling defects, yet it remains unclear whether cilia sequester pathway components to regulate their activation or cilia-associated proteins directly modulate developmental signaling events. To elucidate this question, this study conducted an RNAi-based screen in Drosophila non-ciliated cells to test for cilium-independent loss-of-function phenotypes of ciliary proteins in developmental signaling pathways. The results show no effect on Hedgehog signaling. In contrast, the screen identified several cilia-associated proteins as functioning in canonical Wnt signaling. Further characterization of specific components of Intraflagellar Transport complex A uncovered a cilia-independent function in potentiating Wnt signals by promoting β-catenin/Armadillo activity (Balmer, 2015).

A systematic screen was perfomred to test for potential roles of ciliary proteins in non-ciliated epithelial cells and developmental signaling pathway contexts. This approach identified roles for several cilia-associated proteins in regulating N, Wg, and EGFR signaling, demonstrating cilia-independent functions in vivo in Drosophila. Ciliary proteins have no effect on Hh signaling in non-ciliated cells (Balmer, 2015).

Components of the Hh/Shh pathways are highly conserved between Drosophila and vertebrates, with the main difference being that Shh/Hh signaling takes place in the cilium in vertebrate cells. The lack of a requirement for conserved ciliary proteins in Hh signaling in Drosophila epithelial cells thus suggests that the cilium provides a structural compartment for transducing Hh signals, rather than that specific cilia-associated proteins are required within the pathway. Consistent with this, it was recently demonstrated that Hh-signaling components are localized within the cilium in ciliated Drosophila neuronal cells (Kuzhandaive, 2014). The IFT-B protein IFT25, which has no effect on cilia assembly but is required for Hh signaling in vertebrates, is the exception. However, IFT25 is not conserved in Caenorhabditis elegans or Drosophila. The importance of IFT25 suggests that moving Shh-signaling components along the axoneme (in and out of the cilium) is also critical for Shh signaling in vertebrates, which is again consistent with the notion that Shh signaling needs to take place inside the cilium when cilia are present (Balmer, 2015).

Evolutionary aspects of the Hh/Shh pathway components, studied in planarians, revealed that Hh signaling might have originally been organized by the cilium, with cilia serving as a signaling compartment. The evolutionary loss of IFTs in planaria does not affect Hh signaling, consistent with the notion that the cilium serves as a Hh/Shh signaling hub but that specific components of ciliogenesis are not required for Hh signaling per se (Balmer, 2015).

This screen for developmental signaling requirements of ciliary proteins in non-ciliated cells identified several such factors as being required for canonical Wg/Wnt signaling. Previous studies have proposed conflicting effects of the loss of cilia on canonical Wnt signaling in vertebrates, ranging from reduction or loss of signaling to overactivation of the Wnt pathway. These different, or even opposing, outcomes were possibly caused by pathway crosstalks, context-specific interactions, or both. For example, Shh signaling and Wnt signaling often mutually affect each other, and when cilium biogenesis is impaired, Shh signaling is reduced or abolished. Thus, the potential positive effect of impaired ciliary function on Wnt signaling could be secondary to the loss of Shh signaling, leading to an overactivation of Wnt signaling in ciliary mutants. In particular, mutations in a dynein subunit of the retrograde transport complex (Dync2h1) have been shown to display cell-type-specific effects on cilium integrity and canonical Wnt signaling. The dync2h1 mouse embryonic fibroblasts (MEFs) and regions of dync2h1 mutant embryos that exhibit loss of cilia, and thus loss of Shh signaling, induce overactivation of Wnt signaling. However, in dync2h1 mutant embryos or small interfering RNA in MEFs that disrupt retrograde transport but leave the cilium intact (and thus do not disrupt Shh signaling) display reduced canonical signaling levels (Balmer, 2015).

This study demonstrates that a subset of IFT-A components, as well as other ciliary proteins, act positively in canonical Wnt/Wg signaling in a cilium-independent manner. This is consistent with the preceding data, in particular the effects of the dync2h1 mutants that leave cilia intact. Importantly, these data provide insight into the role of cilia-associated proteins outside of the ciliary structure. This mechanistic study in Drosophila has focused and the IFT-A proteins, and it was demonstrated that a subset of these is required for stabilization, localization, or both of β-cat/Arm prior to its activity in the nucleus. Taken with the previous observations, this non-ciliary function is likely conserved in vertebrates. Due to the omnipresence of primary cilia in vertebrate cells and the associated difficulty of uncoupling ciliary and non-ciliary functions of IFT-A proteins, Drosophila provides a unique and ideal model system to dissect their non-ciliary function. These data also suggest that IFT-A functions in a different configuration in ciliary versus non-ciliary contexts, because IFT144 (although efficiently knocked down) did not affect any detectable aspects of developmental signaling pathways tested. IFT144 is a core structural component of IFT-A in cilia, thus suggesting a different composition of IFT-A outside of the ciliary compartment. However, IFT43 and IFT121, which are described as peripheral within IFT-A in the cilium, show effects (although the IFT121 effects are generally weaker, both in KDs and in mutants, as compared to other IFT-A components), suggesting that some aspects of IFT-A are nonetheless preserved between the cilia and the cytoplasmic locations (Balmer, 2015).

Can these observations be related to disease aspects of the spectrum of ciliopathies? Mutations in human IFT-A components cause a specific subcategory of ciliopathies, called skeletal ciliopathy, which are characterized by limb morphogenesis defects, as well as extra-skeletal abnormalities, including retinal or renal defects. IFT-A mutations often lead to Shh/Hh pathway disruption, which in turn induces skeletal morphogenesis problems similar to the ones observed in skeletal ciliopathies. Importantly, the canonical Wnt-signaling pathway has been proposed to act downstream of Hh and bone morphogenetic protein signaling in bone morphogenesis, and it is therefore possible that defects observed in these syndromes are due to impaired Hh and Wnt signaling. Retinal and renal defects can also be induced by defective Wnt signaling levels. This work supports this concept and adds insight into the function of ciliary proteins with regards to Wnt signaling (Balmer, 2015).

It remains unclear whether the cytoplasmic IFT-A proteins (in their possibly altered configuration) associate with microtubular structures and whether such an association is required for function in Wnt/Wg signaling. Interestingly, a role for kinesin II in Wg signaling and the transport of *beta:-cat/Arm was recently reported (Vuong, 2014), consistent with a link to microtubules. One hypothesis is that the IFT-A proteins associate with microtubules and play a similar role to Costal-2 (Cos-2)/Kif7 in Hh signaling. In the absence of Hh signals, Cos-2 binds to the Hh/Shh effector Ci/Gli and tethers it to microtubules, together with Fused, Suppressor of Fused, and several kinases. Hh activation reverses this binding and frees Ci from the complex. Cos-2 is a negative regulator of Hh signaling, and the current data suggest that IFT-A proteins act antagonistically to the destruction complex by promoting β-cat/Arm stabilization. Sequestering Arm away from the destruction complex to prevent its phosphorylation and allow activation of the pathway makes this an attractive hypothesis for future study (Balmer, 2015).

Larval

In the larval brain, ARM and Actin are enriched in fiber tracts of the ventral ganglion and the optic lobes. In the developing eye, ARM is uniformly distributed in front of the morphogenetic furrow, but is non-randomly distributed after the passage of the furrow. In the furrow ARM protein accumulates in a star-shaped pattern on membranes where cells in each pre-cluster abut one another but not on membranes separating the clusters from undifferentiated cells. Actin remains more diffuse until well after the passing of the furrow (Peifer, 1990).

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).

A study of APC1 and APC2 examines asymmetric protein localization in larval neuroblasts

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).

Arm function in the wing imaginal disc

In the third thoracic segment of Drosophila, wing development is suppressed by the homeotic selector gene Ultrabithorax (Ubx) in order to mediate haltere development. Ubx represses dorsoventral (DV) signaling to specify haltere fate. The mechanism of Ubx-mediated downregulation of DV signaling has been studied. Wingless (Wg) and Vestigial (Vg) are differentially regulated in wing and haltere discs. In wing discs, although Vg expression in non-DV cells is dependent on DV boundary function of Wg, it maintains its expression by autoregulation. Thus, overexpression of Vg in non-DV cells can bypass the requirement for Wg signaling from the DV boundary. Ubx functions, at least, at two levels to repress Vestigial expression in non-DV cells of haltere discs. At the DV boundary, it functions downstream of Shaggy/GSK3ß to enhance the degradation of Armadillo (Arm), which causes downregulation of Wg signaling. In non-DV cells, Ubx inhibits event(s) downstream of Arm, but upstream of Vg autoregulation. Repression of Vg at multiple levels appears to be crucial for Ubx-mediated specification of the haltere fate. Overexpression of Vg in haltere discs is enough to override Ubx function and cause haltere-to-wing homeotic transformations (Prasad, 2003).

Several experiments were designed to test the current model of Wg and Vg regulation (which is essentially based on studies on wing imaginal discs) in haltere discs. In wing discs, both Wg and Vg are subjected to an elaborate regulatory circuit. Wg and Vg interact to maintain each other's expression at the DV boundary. Vg-mediated activation of Wg is independent of Arm and TCF/pan function, which suggests that Vg may activate Wg either directly or through the N signaling pathway. Vg is capable of specifying wing development, even in the absence of Wg signaling. Overexpression of Vg in a vg1/vg1 background (in which no Wg or Vg is expressed) is sufficient to rescue wing phenotypes. This is particularly significant because Vg was expressed in this experiment only in non-DV cells. These results also suggest that Vg cell-autonomously regulates its own expression through its quadrant enhancer. Clonal analysis of arm suggests that Wg is required to activate vg-QE and Arm is not able to activate this enhancer in vg1 background. Wg signaling might activate Vg either indirectly or by activating some other enhancer of Vg. Once activated, Vg might maintain its expression by autoregulation, which is mediated through its quadrant enhancer. This could ensure the maintenance of Vg expression in non-DV cells, once it is activated by Wg signaling. It might also explain how the Wg gradient is translated into uniformly higher levels of Vg in non-DV cells (Prasad, 2003).

However, the above-mentioned model does not reconcile the observation that Vg, and not Wg, is capable of activating vg-QE in Ser background. Since the vg gene is intact in Ser background, ectopic expression of Wg using dpp-GAL4 should have activated one of the enhancers to induce Vg expression, which in turn would activate vg-QE. A model that reconciles all the results would, therefore, include a third component, which may act either parallel to or downstream of Wg and Vg at the DV boundary. Although there is no direct evidence for the existence of such a molecule, the fact that N23-GAL4 expression in non-DV cells is dependent on N function and independent of Vg and Wg function suggests such a possibility (Prasad, 2003).

The downregulation of Wg signaling by Ubx occurs at the level of Arm stabilization. Ubx inhibits stabilization of Arm by acting on event(s) downstream of Sgg. Normally, the Arm degradation machinery is very efficient and can degrade even overexpressed Arm. This is evident from the fact that embryos overexpressing Arm (from armS2) secrete normal denticle belts. If a downstream component functions with enhanced efficiency (either by direct enhancement of its expression by Ubx or owing to repression of a positive component of Wg signaling), residual activity of Sgg may be sufficient to cause enhanced degradation of Arm. Thus, enhanced degradation of Arm in haltere discs provides a new assay system to identify additional components of Wg signaling. For example, in microarray experiments to identify genes that are differentially expressed in wing and haltere discs, several transcripts of known (e.g., Casein kinase) and putative (e.g., Ubiquitin ligase) negative regulators of Wg signaling are upregulated in haltere discs (Prasad, 2003).

The results suggest that Wg and Vg regulation in haltere discs is different from that in wing discs. Wg is not autoregulated in haltere discs. In addition, Vg expression at the haltere DV boundary is independent of Wg function. However, in both wing and haltere discs, Wg expression at the DV boundary is dependent on Vg. Wg expression at the anterior DV boundary of haltere discs could be redundant because overexpression of DN-TCF at the haltere DV boundary shows no phenotype. However, Vg at the DV boundary appears to have an independent function. vg1 flies exhibit much smaller halteres than do wild-type flies. Since Wg function (and expression in the posterior compartment) is already repressed in haltere discs, reduction in haltere size in vg1 flies suggests Wg-independent long-range effects of Vg from the DV boundary. This could be one of the reasons why Ubx does not affect Vg expression at the DV boundary but represses Vg expression in non-DV cells. In wing discs too, Vg may have such a function on cells at a distance (Prasad, 2003).

One way to test the requirement of Ubx in DV and non-DV cells directly is by removing Ubx only from the haltere DV boundary or from non-DV cells. Clonal removal of Ubx solely from the haltere DV boundary does not induce cuticle phenotype in the capitellum. However, it was not possible to ascertain the effect on vg-QE because of haploinsufficiency: Ubx-heterozygous haltere discs themselves show activation of lacZ in the entire haltere pouch. The activation of vg-QE in Ubx/+ haltere discs could be a result of reduced Ubx function at the DV boundary, or in non-DV cells, or in both. Misexpression of Ubx at the wing disc DV boundary causes non-cell-autonomous reduction in vg-QE expression. The current results suggest that Ubx represses additional event(s) in non-DV cells to downregulate Vg expression. This is consistent with the recent report on cell-autonomous repression of vg-QE by ectopic Ubx in wing discs. It is proposed that Ubx inhibits the activation of Vg in non-DV cells at three different levels: (1) Wg in the posterior compartment; (2) event(s) downstream of Sgg that inhibit the stabilization of Arm, and (3) additional event(s) downstream of Arm in non-DV cells. In wing discs, Wg and a hitherto unknown DV component may function together to activate Vg in non-DV cells. Since Vg-autoregulation is not inhibited in haltere discs, it is possible that Ubx represses Vg activation in non-DV cells by interfering with the Wg-mediated activation of Vg and/or by repressing the activity of the unknown DV-signal molecule in the haltere (Prasad, 2003).

The cellular events that govern patterning during animal development must be precisely regulated. This is achieved by extrinsic factors and through the action of both positive and negative feedback loops. Wnt/Wg signals are crucial across species in many developmental patterning events. Drosophila nemo (nmo) acts as an intracellular feedback inhibitor of Wingless (Wg) and it is a novel Wg target gene. Nemo antagonizes the activity of the Wg signal, as evidenced by the finding that reduction of nmo rescues the phenotypic defects induced by misexpression of various Wg pathway components. In addition, the activation of Wg-dependent gene expression is suppressed in wing discs ectopically expressing nmo and enhanced cell autonomously in nmo mutant clones. nmo itself is a target of Wg signaling in the imaginal wing disc. nmo expression is induced upon high levels of Wg signaling and can be inhibited by interfering with Wg signaling. Finally, alterations are observed in Arm stabilization upon modulation of Nemo. These observations suggest that the patterning mechanism governed by Wg involves a negative feedback circuit in which Wg induces expression of its own antagonist Nemo (Zeng, 2004).

In Drosophila, several examples of Wg feedback inhibition have been identified. (1) It has been shown that Wg downregulates its own transcription in the wing pouch to narrow the RNA expression domain at the DV boundary. (2) Wg signaling can repress the expression of its receptor Dfz2 in the wg-expressing cells of the wing disc. Wg regulation of Dfz2 creates a negative feedback loop in which newly secreted Wg is stabilized only once it moves away from the DV boundary to cells expressing higher levels of Drosophila Fz2. (3) The Wg target gene naked cuticle (nkd) acts through Dsh to limit Wg activity. (4) Wingful (Wf), an extracellular inhibitor of Wg, is itself induced by Wg signaling (Zeng, 2004).

This research adds Nemo to this list of inducible antagonists participating in Wg signaling. Nemo antagonizes the Wg signal in wing development, as evidenced by phenotypic rescue, suppression of Wg-dependent gene expression in discs ectopically expressing nmo, and ectopic expression of a Wg-dependent gene in nmo mutant clones (Zeng, 2004).

In support that Wg signaling regulates the transcription of nmo, several dTCF consensus binding sites have been found in the 5' region of the nmo gene that may represent enhancer elements. Indeed, two sites match 9 out of 11 bp (GCCTTTGAT) of the T1 site (GCCTTTGATCT) in the dpp BE enhancer that has been shown both in vitro and in vivo to bind and respond to dTCF. The presence of these sites suggests that the observed transcriptional regulation of nmo by Wg may involve direct binding to the nmo DNA sequence by dTCF (Zeng, 2004).

As a result of comparing the endogenous expression pattern of nmo with stabilized Arm, it was noticed that the highest levels of Nemo exclude Arm stabilization, while high levels of Arm are present in cells in which nmo levels are lower. Since Arm protein stabilization is a direct consequence of Wg pathway activation, attempts were made to examine whether Nemo may function to inhibit Wg by promoting Arm destabilization and subsequent breakdown. Indeed, ectopic expression of Nemo can lead to cell-autonomous reduction in Arm protein levels. This preliminary result suggests a mechanism in which Nemo may contribute to the destabilization of Arm that involves the Axin/APC/GSK3 complex. One explanation to account for such a finding would concern the interaction with TCF in the nucleus and the role of dTCF as an anchor for Arm. Given what is known about NLKs, it is likely that Nemo may act on the ability of the dTCF/Arm complex to bind DNA and activate transcription. It has been proposed that dTCF acts as an anchor for Arm in the nucleus. It remains to be determined how efficient this anchor is and whether there are conditions in which the interaction may become compromised, such as is seen with elevated Nemo. NLKs have been shown to affect the DNA-binding ability of TCF/ß-catenin. Perhaps in the absence of DNA binding, this complex is less stable and Arm could be free to shuttle to the cytoplasm where it could associate with Axin or APC and become degraded. It is proposed that the ectopic nmo leads to destabilization of the dTCF/Arm/DNA complex, thus causing Arm to exit the nucleus and be degraded through interaction with Axin, APC and GSK3. The observation that ectopic expression of full-length Arm cannot induce any activated Wg phenotypes has been explained by the hypothesis that even these high levels of protein are not sufficient to overcome the degradation machinery. Thus, the finding that there is no elevated Arm in nmo clones is consistent with an inability to overcome the endogenous degradation machinery; even though less Nemo could lead to more stabilized DNA interactions, this would not lead to higher levels of stabilized Arm than are normally found (Zeng, 2004).

Invasive cell behavior during Drosophila imaginal disc eversion is mediated by the JNK signaling cascade

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).

Fat and Wingless signaling oppositely regulate epithelial cell-cell adhesion and distal wing development

Development of organ-specific size and shape demands tight coordination between tissue growth and cell-cell adhesion. Dynamic regulation of cell adhesion proteins thus plays an important role during organogenesis. In Drosophila, the homophilic cell adhesion protein DE-Cadherin regulates epithelial cell-cell adhesion at adherens junctions (AJs). This study shows that along the proximodistal (PD) axis of the developing wing epithelium, apical cell shapes and expression of DE-Cad are graded in response to Wingless, a morphogen secreted from the dorsoventral (DV) organizer in distal wing, suggesting a PD gradient of cell-cell adhesion. The Fat (Ft) tumor suppressor, by contrast, represses DE-Cad expression. In genetic tests, ft behaves as a suppressor of Wg signaling. Cytoplasmic pool of ß-catenin/Arm, the intracellular transducer of Wg signaling, is negatively correlated with the activity of Ft. Moreover, unlike that of Wg, signaling by Ft negatively regulates the expression of Distalless (Dll) and Vestigial (Vg). Finally, Ft is shown to intersect Wnt/Wg signaling, downstream of the Wg ligand. Fat and Wg signaling thus exert opposing regulation to coordinate cell-cell adhesion and patterning along the PD axis of Drosophila wing (Jaiswal, 2006).

Cells of the dorsoventral (DV) boundary in the wing imaginal disc synthesize Wg. The DV boundary marks the distal end of the growing appendage, while the future hinge region, displaying Wg expression in two concentric rings, marks the proximal wing. The lacZ reporter of the quadrant enhancer of vestigial (vg), Q-vg-lacZ marks the entire distal wing [i.e. the presumptive wing blade (pouch) (Jaiswal, 2006).

In optical sections of the imaginal disc epithelium, AJs are visualized in the XY or XZ planes based on immunolocalization of DE-Cad and ß-catenin/Arm, besides binding with fluorochrome conjugated Phalloidin to F-actin. Both ß-catenin/Arm and DE-Cad display characteristic upregulation across the DV boundary along the PD axis of the wing imaginal disc. Optical sections along the XY plane reveal higher levels of DE-Cad localization and narrower apical circumferences in the AJs of cells flanking the DV boundary when compared with those of the more proximally located cells. Optical sections along the XZ plane further confirmed upregulation of DE-Cad. Thus, along the PD axis of the wing disc, cell shapes and DE-Cad levels are graded (Jaiswal, 2006).

Whether the PD gradient of cell shape and DE-Cad levels are linked to Wg signaling was tested. Somatic clones displaying constitutive Wg signaling (induced by overexpression of Dsh or of a degradation resistant variant of ß-catenin/Arm, ArmS10) induce cell-autonomous upregulation in the levels of DE-Cad and apical cell constrictions. Somatic clones expressing secreted Wg, however, are expected to induce non-cell-autonomous effects. Indeed, these clones induced non-cell autonomous and graded upregulation in the levels of DE-Cad in the AJs and changes in apical cell shapes. In the presumptive hinge region, Wg overexpression produces a more striking pattern of non-cell autonomous changes in cell shapes: cells neighboring the Wg-expressing cells appear to organize as whorls around the former and display epithelial misfolding (Jaiswal, 2006).

Furthermore, expression of GPI-anchored DFz2 receptor GPI-DFz2, which compromises Wg signaling, obliterates the characteristic PD gradient in the levels of DE-Cad and F-actin in the AJs. Finally, loss of Wg expression in the DV boundary of wing imaginal disc of Nts mutants grown at a restricted temperature also abolishes the PD gradient of DE-Cad and apical cell shapes. To further test if apical cell constrictions are linked to elevated levels of DE-Cad in AJs, DE-Cad was expressed in somatic clones. These clones were apically constricted, consistent with the role of DE-Cad/E-Cad in remodeling cell shape and tissue architecture. These results thus link Wg signaling to the PD gradient in the levels of DE-Cad and apical cell shapes in the wing imaginal discs (Jaiswal, 2006).

Somatic clones with altered cell-cell adhesion sort out from their neighbors and display smooth clone borders. Indeed, somatic clones displaying gain of Wg signaling owing to Dsh or ArmS10 misexpression sort out from their neighbors and display smooth clone borders, akin to those misexpressing DE-Cad. Wg signaling may alter cell-cell adhesion by enhancing recruitment of ß-catenin/Arm to the AJs and/or by its transcriptional input. In many cell types, for example, expression of cadherins rather than the levels of catenins appears to be the rate-limiting step of Catenin-Cadherin complex formation at AJs and cell-cell adhesion. Wild type ß-catenin/Arm (ArmS2), when overexpressed, does not transduce Wg signaling. Somatic clones overexpressing ArmS2 display 'wiggly' clone borders, unlike those expressing Dsh or ArmS10. Thus, expression of ß-catenin/Arm alone, without a concomitant enhancement of Wg signaling, fails to alter cell-cell adhesion. Cell-cell adhesion in wing imaginal disc epithelium is therefore likely to be regulated by transcriptional input from Wg signaling (Jaiswal, 2006).

To test if canonical Wg signaling regulates DE-Cad expression, the response of its lacZ reporter, DE-Cad-lacZ, was examined. Cells receiving high threshold of Wg signaling in the wing imaginal discs, as in those flanking the DV boundary, displayed higher levels of DE-Cad-lacZ reporter activity when compared with those further away from the source of Wg expression. Furthermore, somatic clones expressing ArmS10 or Dsh display cell-autonomous activation of the DE-Cad-lacZ. Finally, clones expressing the secreted Wg induce non-cell-autonomous activation of DE-Cad-lacZ: i.e., in cells within and surrounding the clones. Together, these results suggest that regulation of DE-Cad by the long-range activity of the Wg morphogen sets up the PD gradient of cell-cell adhesion and cell shape in the distal wing (Jaiswal, 2006).

Somatic clones lacking Ft (ft-/ft-), marked by loss of GFP, display overgrowth and altered cell-cell adhesion with characteristic circular and smooth clone borders, unlike the 'wiggly' borders of their wild type (ft+/ft+) twins that are marked by brighter GFP. Furthermore, cells lacking Ft displayed upregulation of DE-Cad in their AJs and DE-Cad-lacZ. By contrast, when Ft was overexpressed, levels of both DE-Cad or DE-Cad-lacZ were downregulated. Besides, following overexpression of Ft in the posterior wing compartment, cells flanking the DV boundary displayed wider apical circumferences when compared with those of the anterior wing compartment. These results suggest that Ft regulates DE-Cad expression, cell-cell adhesion and apical cell shapes in the distal wing (Jaiswal, 2006).

The results suggest that by regulating DE-Cad expression, Wg signaling integrates cell-cell adhesion with tissue growth and pattern. Regulation of DE-Cad expression could be a prevalent mechanism for coordination of the emerging pattern in an organ primordium with the spatial control of its cell-cell adhesion. For example, DE-Cad levels are also upregulated in cells flanking the stripe of cells along the AP boundary that express the morphogen Decapentaplegic (Dpp); misregulation of Dpp signaling also affects DE-Cad expression. The Ft tumor suppressor, by contrast, negatively regulates DE-Cad expression in the distal wing. This may also explain the inverse correlation between the levels of DE-Cad in AJs and the activity of Ft. Thus, besides its heterophilic binding with Ds, Ft controls cell-cell adhesions at AJs by regulating DE-Cad expression (Jaiswal, 2006).

Apart from cell-cell adhesion, DE/E-Cad regulation may impact a variety of other cellular processes and developmental mechanisms. E-Cad has been shown to mark the sites of actin assembly on cell surface. Cadherin complexes regulate cytoskeletal networks and cell polarity, while disruption of AJ associated components affects asymmetric cell division. Fat1, a mammalian homolog of Drosophila Ft, modulates actin dynamics. Interestingly, Ft also regulates orientated cell division (OCD) in imaginal epithelium, which is mirrored by orientation of the spindles of the dividing cells; OCD may also regulate organ shape along the PD axis. Misregulation of DE-Cad may thus affect the cytoskeleton and produce OCD phenotype in ft mutant discs (Jaiswal, 2006).

In both loss- and gain-of-function assays, this study shows that Ft downregulates Dll and Vg/Q-vg-lacZ in the distal wing. Although Vg/Q-vg-lacZ and Dll have not been ascertained to be the direct targets of Wg, all available evidence so far suggests that these targets positively respond to Wg signaling. These results also show that Ft and Wg signaling intersect and control distal wing growth and pattern, presumably through their opposing regulation of a common set of targets, namely, DE-Cad, Vg and Dll. Apart from Wg signaling, Dpp signaling also regulates Q-vg-lacZ; however, its long-range target, Omb is not upregulated in ft mutant clones, suggesting that regulation of distal wing targets by Ft is mediated by its intersection with Wg signaling (Jaiswal, 2006).

The results show that Ft negatively regulates Wg signaling. Loss or gain of Ft induces a telltale sign of perturbations in Wg signaling, namely, changes in the cellular pool of ß-catenin/Arm, consistent with its role as a suppressor of Wg signaling in genetic tests. The results further reveal intersection of Ft with Wg signaling downstream of the Wg ligand, while with respect to its receptor, Ft is likely to act either upstream of or parallel to Fz/Fz2. It is interesting to note here that the role of Ft in PCP regulation has also been suggested to be either parallel to or upstream of the Fz receptor. It is also noted that Ft co-localizes with neither Fz nor Fz2 and does not mediate their subcellular localization, thereby suggesting that Ft interacts with Fz indirectly. Unraveling the genetic and molecular basis of this interaction may explain how Ft straddles both the canonical (growth and cell-cell adhesion) and non-canonical (PCP; see Eisenmann's Wnt Signaling) Wnt signaling pathways (Jaiswal, 2006).

One of the remarkable aspects of development of an organ primordium is that a stereotypic PCP is achieved even while it passes through dynamic changes in its size and shape. The fact that changing organ sizes/shapes does not alter PCP suggests an in-built mechanism to regulate constancy of PCP during animal development. A link between PCP and growth through the activity of Ft has been speculated, since it regulates both. Intersection of Ft and the canonical Wg signaling seen here might provide a mechanism to coordinate PCP and organ growth (Jaiswal, 2006).

Drosophila wing growth is under dynamic spatial and temporal regulation by Wg signaling. Furthermore, different thresholds of Wg signaling impact cell proliferation in their characteristic ways and activate distinct sets of PD markers. Although at a very high threshold, Wg signaling inhibits cell proliferation, at a modest threshold it has been shown to stimulate growth. It is noted that loss of Ft fails to activate Wg targets that demand a high threshold of Wg signaling, e.g., Ac, which is required for wing margin specific bristle development. Conversely, overexpression of Ft also does not lead to loss of margin bristles, suggesting that it is not a strong repressor of Wg signaling either. The short-range Wg target, fz3-lacZ, which responds to a high threshold of Wg signaling, is also not upregulated by loss of Ft. Dll responds to a higher threshold of Wg signaling than that required for Vg/Q-vg. Dll and Vg display modest and strong upregulation respectively, following loss of Ft. These results suggest that loss of Ft upregulates Wg signaling to only modest thresholds, consistent with the growth-promoting effect of the latter (Jaiswal, 2006).

Over-proliferation in ft mutant imaginal discs is induced by perturbation of as yet unidentified disc-intrinsic mechanisms that determine the discs' characteristic final sizes. The imaginal discs of ft mutants continue to grow and the extent of their over-proliferation appears to be constrained only by the developmental time available during the extended periods of their larval life. By contrast, growth in wild-type imaginal discs is determinate, which ceases after they attain their predetermined sizes even under conditions of unlimited developmental time; for example, on transplantation into wild-type adult host abdomen that can sustain development. ft mutant imaginal discs thus acquire unlimited proliferative potential, akin to immortalization, a crucial step during tumorigenesis. It is significant that the Ft tumor suppressor downregulates Wg/Wnt signaling, a pathway implicated in cancers. Several orthologs of Ft have been identified in vertebrates with diverse functions. It will thus be interesting to explore if these orthologs of Ft in higher vertebrates also interact with Wnt signaling and thereby behave as tumor suppressors (Jaiswal, 2006).

Wingless signaling modulates cadherin-mediated cell adhesion in Drosophila imaginal disc cells

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).

Tissue remodeling during maturation of the Drosophila wing

The final step in morphogenesis of the adult fly is wing maturation, a process not well understood at the cellular level due to the impermeable and refractive nature of cuticle synthesized some 30 h prior to eclosion from the pupal case. Advances in GFP technology now make it possible to visualize cells using fluorescence after cuticle synthesis is complete. Between eclosion and wing expansion, the epithelia within the folded wing begin to delaminate from the cuticle and that delamination is complete when the wing has fully expanded. After expansion, epithelial cells lose contact with each other, adherens junctions are disrupted, and nuclei become pycnotic. The cells then change shape, elongate, and migrate from the wing into the thorax. During wing maturation, the Timp gene product, tissue inhibitor of metalloproteinases, and probably other components of an extracellular matrix are expressed that bond the dorsal and ventral cuticular surfaces of the wing following migration of the cells. These steps are dissected using the batone and Timp genes and ectopic expression of αPS integrin, inhibitors of Armadillo/β-catenin nuclear activity and baculovirus caspase inhibitor p35. It is concluded that an epithelial-mesenchymal transition is responsible for epithelial delamination and dissolution (Kiger, 2007; full text of article).

The following outline is proposed of that program based upon cell behavior: delamination and severing contacts; changing cell shape; and migration and ECM synthesis.

Stage 1, delamination and severing contacts

A signaling role for integrins during the prepupal apposition has been proposed that prepares cells for integrin-based adhesion of the epithelia at the pupal apposition. The observation that wing epithelial cells persist in the blistered regions produced by ectopic αPS integrin expression suggests that the integrin interaction also prepares cells to respond to the later signal that induces epithelial delamination and dissolution. This signal is also blocked in the mutant batone, which prevents wing expansion. Some cells begin to delaminate from the cuticle before wing expansion has begun, and all have delaminated by the time expansion is complete. Delamination must involve severing of ECM contacts. The precision of the cellular array in a newly open wing must derive from cell–cell contacts between stretched cells that are maintained following delamination. Each cell then compacts and becomes round (as judged by the increase in fluorescence intensity). The round cells have evidently severed their junctions with adjacent cells because the precise array of cells begins to break up and Arm-GFP moves from the cell membrane to the cytoplasm (Kiger, 2007).

It would appear that disturbing the normal state of Arm/β-catenin signaling activity in epithelial cells blocks delamination. Delamination is blocked by ectopic expression of Pygo in the epithelial cells, which blocks expression of Arm target genes in a variety of tissues, and by ectopic expression of Shaggy, which blocks expression of Arm target genes by phosphorylating cytoplasmic Arm, promoting its degradation and depleting nuclear Arm. Ectopic expression of stabilized forms of Arm not subject to Shaggy phosphorylation evidently has a dominant-negative effect on Arm signaling activity in the maturing wing, blocking delamination of epithelial cells. This interpretation is supported by the following observations. First, no effect is produced by ectopic expression of wild-type Arm using the same Gal4-30A driver, consistent with other reports, very likely indicating the efficiency with which wild-type Arm is eliminated by phosphorylation and degradation through the proteasome. Second, a very low level of nuclear Arm is sufficient for target gene expression. The Arm-GFP fusion protein used here is fully active and completely covers homozygosity for a null arm allele, yet nuclear Arm-GFP cannot be detected in cells receiving a Wingless signal. Thus, it is reasonable that non-physiologically high levels of stable forms of Arm could have a dominant-negative effect, not unlike the inhibitory effect of over-expression of Pygo on Arm-directed transcription. (Kiger, 2007).

Arguing against an interpretation that the effects of ectopic gene expression might be non-specific, note that Gal4-30A-driven expression of p35 does not block delamination. Nor does Gal4-30A-driven expression of either αPS integrin or wild-type transcription factor Pangolin/dTCF/LEF-1, or a dominant-negative form of CREB have any effect on wing maturation (Kiger, 2007).

Stage 2, changing cell shape

The round cells then begin to change shape, extending thin cytoplasmic filaments, and elongate into spindles that associate with similarly shaped cells forming streams. The fact that p35 expression interrupts developmental progression at the round cell stage clearly separates Stage 1 from the changes in cell shape, cell migration, and ECM synthesis events that follow. In some cellular contexts caspase inhibition prevents cell migration independently of blocking apoptosis. It has been shown that the nuclei of wing cells cease to retain nuclear-targeted GFP and begin to fragment their DNA at what appears to be the round cell stage, consistent with the observation of pycnotic nuclei at this stage (Kiger, 2007).

Stage 3, migration and ECM synthesis

The cells migrate toward the hinge and into the body of the fly, leaving behind components, perhaps including tissue inhibitor of metalloproteinases, of an ECM that will bond dorsal and ventral cuticular surfaces. It is noteworthy that Timp deficiency does not interfere with cell migration. ECM assembly must be the final step in the developmental program. The nonautonomous action of Timp in bonding cuticle secreted by mutant Timp clones suggests that Timp is present in abundance and diffuses over large distances in the wing to participate in ECM formation (Kiger, 2007).

Precisely how ectopic expression of the various UAS transgenes studied in this paper produces wing blisters or collapsed wings is not wholly clear. It seems doubtful that cells that fail to delaminate during early phases of tissue remodeling would secrete ECM components normally. Yet a variable number of cells in these wings do delaminate and leave the wing, presumably because of variation in the level of Gal4-30A expression. These cells might be expected to secrete the necessary ECM components, although the level of critical component(s) may be insufficient for normal bonding to occur in some cases. Blister formation might also be caused by the presence of numbers of undelaminated cells physically preventing ECM from bonding the underlying cuticle. Note that when ectopic p35 expression is limited, a moderate number of round, delaminated cells can become bound in the wing without producing blisters (Kiger, 2007).

The presence of true hemocytes in the wing raises the question of whether these cells play a role in wing maturation. If Gal4-30A was to be expressed in these cells, as well as in epithelial cells, interpretation of ectopic expression studies would be complicated. No cells were detected expressing DsRedGFP fluorescence that did not express ywing-GFP fluorescence, suggesting that Gal4-30A is not expressed in true hemocytes. The observations that Hemese-Gal4-driven expression of Shaggy or of Pygo has no effect on wing maturation strongly suggest that the effects of Shaggy and Pygo on wing maturation are not mediated by true hemocytes exclusively, if at all. While the possibility that Timp and/or other ECM components are supplied by true hemocytes cannot be ruled out, the bulk of the evidence supports an active role for epithelial cells in bonding the wing surfaces. Precocious death of epithelial cells induced by Gal4-30A-driven expression of Ricin A in late pupal epithelial cells prevents bonding of dorsal and ventral cuticle after eclosion. Because the wing cuticle is fully formed, the induced cell death must have occurred after cuticle deposition but before eclosion. UV irradiation after eclosion blocks both epithelial cell delamination and bonding of the wing surfaces. In addition, it is clear that mitotic clones of defective epithelial cells affect bonding of the wing surfaces. Mitotic clones mutant for an integrin gene produce blisters in the wing cuticle as do mitotic clones ectopically expressing PKAc (Kiger, 2007).

These studies describe for the first time the developmental program that completes morphogenesis of the adult fly. The requirement for a normal state of Arm/β-catenin signaling activity suggests that an epithelial–mesenchymal transition (EMT) transforms epithelial cells into mobile fibroblasts in the wing (Kiger, 2007).

The best known example of an EMT in Drosophila is neuroblast delamination. In embryonic central nervous system formation, Wingless signaling has been shown to induce nonautonomously the delamination of specific neuroectoderm cells to form S2 neuroblasts. In peripheral nervous system formation, Wingless signaling is required for bristle formation at the wing margin, and ectopic expression of Wingless induces ectopic bristles in the wing blade. The ability of Wingless to induce neuroectoderm cells to form neuroblasts is tightly regulated by Notch in both the central and peripheral nervous systems. Evidence supports the idea that Notch modulates Wingless signaling by associating directly with Arm/β-catenin to regulate its transcriptional activity (Kiger, 2007).

Arm/β-catenin signaling appears to be characteristic of EMTs. Translocation of Arm/β-catenin into the nucleus precedes gastrulation in Drosophila, the sea urchin, and zebrafish. EMTs occur in the vertebrate neural crest when cells delaminate from the neural epithelium and migrate throughout the embryo. In the avian neural crest, dominant-negative forms of β-catenin and LEF/TCF inhibit delamination of cells from the epithelium, G1/S transition, and transcription of target genes. β-Catenin and LEF/TCF proteins are observed to translocate to the nuclei of avian neural crest cells only during delamination and to be absent during advanced stages of migration. EMTs are also a characteristic of cancer formation and can be initiated in some cancers by aberrant β-catenin activity (Kiger, 2007).

Multiple ways of activating Arm/β-catenin signaling exist. There are two independently regulated pathways that can target Arm/β-catenin to the proteasome, the Shaggy/Glycogen synthase kinase 3 degradation complex and the Seven in Absentia Homologue/ubiquitin ligase degradation complex. Multiple G-protein-coupled receptors target the Shaggy/Glycogen synthase kinase 3 degradation complex for inhibition. Further studies are necessary to identify the hormone(s), receptor(s) and signal transduction mechanisms acting in the wing maturation program and to relate this work to the extensive studies of the hormonal signals controlling wing expansion and cuticle tanning (Kiger, 2007).

Robustness and stability of the gene regulatory network involved in DV boundary formation in the Drosophila wing: Cut blocks the Wg signaling pathway at the level or upstream of Arm

Gene regulatory networks have been conserved during evolution. The Drosophila wing and the vertebrate hindbrain share the gene network involved in the establishment of the boundary between dorsal and ventral compartments in the wing and adjacent rhombomeres in the hindbrain. A positive feedback-loop between boundary and non-boundary cells and mediated by the activities of Notch and Wingless/Wnt-1 leads to the establishment of a Notch dependent organizer at the boundary. By means of a Systems Biology approach that combines mathematical modeling and both in silico and in vivo experiments in the Drosophila wing primordium, this regulatory network was modeled and tested; evidence is presented that a novel property, namely refractoriness to the Wingless signaling molecule, is required in boundary cells for the formation of a stable dorsal-ventral boundary. This new property has been validated in vivo, promotes mutually exclusive domains of Notch and Wingless activities and confers stability to the dorsal-ventral boundary. A robustness analysis of the regulatory network complements the results and ensures its biological plausibility (Buceta, 2007).

In silico evidence is presented that refractoriness to the Wg signal in boundary cells provides stability to the gene regulatory network. Boundary cells are characterized by high levels of Notch activity, thus suggesting Notch is responsible for making boundary cells refractory to the Wg signal. The role of Notch in this process was analyzed in the developing wing primordium. Ectopic activation of Notch in non-boundary cells represses Wg target gene expression. Note that Notch, in this case, causes ectopic Wg expression in non-boundary cells, which induces target gene expression only in Wg non-expressing cells. By contrast, ectopic expression of Wg alone induces the expression of target genes in both Wg-expressing and non-expressing cells. When boundary cells lack Notch activity, either by mutation or by expression of a dominant negative form of Delta known to titrate out the Notch receptor, these cells start to express target genes of Wg. It can then be concluded that either Notch activity itself, or one or several of its target genes inhibits the expression of Wg target genes in boundary cells (Buceta, 2007).

High levels of Notch activity induce expression of the homeobox gene cut in boundary cells and Cut has been previously shown to be required to repress Delta and Serrate expression in these cells. Then, whether Cut mediates the activity of Notch in inhibiting the expression of other Wg target genes was examined. In the absence of Cut activity, either in a homozygous mutant background or in clones of mutant cells, boundary cells start expressing genes regulated by the Wg signal, and ectopic Notch activation in non-boundary cells is now unable to repress Wg target gene expression. Note that Notch, in this case, causes ectopic expression of Wg, which induces target gene expression in both Wg-expressing and non-expressing cells. Finally, forced expression of Cut in non-boundary cells represses the expression of Wg target genes. Taken together, these results indicate that Cut is not only required but also sufficient to inhibit Wg target gene expression in boundary cells downstream of Notch (Buceta, 2007).

Cut might exert its function either by blocking the Wg signaling pathway or, alternatively, by inhibiting the expression of every Wg target gene. The Wg signaling pathway is activated by controlling the levels and subcellular localization of the transcriptional co-activator Armadillo (Arm, known as β-catenin in vertebrates). In the absence of Wg signal, Arm levels are kept low through degradation. This degradation depends on the phosphorylation of Arm by the kinase Shaggy/Zeste white-3/Glycogen synthase kinase-3β (GSK-3β). Phosphorylated Arm is recognized rapidly by the proteasome and destroyed. Following Wg ligand binding, this degradation is inhibited, which enables Arm to accumulate, enter the nucleus and activate a transcriptional response. In the Drosophila wing, Arm protein levels are severely reduced in boundary cells, when compared with adjacent cells, even though extracellular Wg protein is available in both types of cells. This observation indicates that the activity of the Wg signaling pathway is repressed in these cells at the level or upstream of Arm. Consistent with this observation, a dominantly activated form of Arm (ArmS10), which lacks the GSK-3ß phosphorylation sites and escapes degradation, induces expression of Wg targets in boundary cells. Overexpression of any other limiting factor of the Wg pathway that acts upstream of Arm is unable to induce Wg target gene expression in these cells (Buceta, 2007).

Cut appears to mediate this type of repression of the Wg signaling pathway. In the absence of Cut activity, Arm protein levels are not reduced in boundary cells, and ectopic expression of Cut in non-boundary cells reduces Arm protein levels and represses the expression of Wg target genes. Moreover, ArmS10 can bypass the effects of ectopic Cut expression and restores Wg target gene expression in non-boundary cells. Co-expression of limiting factors of the Wg pathway acting upstream of Arm does not cause this effect. Taken together, these results indicate that Cut blocks the Wg signaling pathway at the level or upstream of Arm. Cut might exert its function through transcriptional regulation of a gene product involved in regulating the degradation of Arm (Buceta, 2007).

So far in vivo evidence has been provided that Cut is required in boundary cells to repress the Wg signaling pathway and also, by means of in silico experiments, it has been shown that such repression leads to a stable DV boundary formation. In silico implementation of the refractoriness to the Wg signal via Cut leads to stable DV boundary formation. The stationary pattern of gene expression and activity observed in this case is in agreement with in vivo results (Buceta, 2007).

The conclusions can be extended further with regard to the role played by Cut in DV boundary formation. In the absence of refractoriness to the Wg signal (provided by the activity of Cut in boundary cells) an initial increase in Notch activity and Wg expression takes place. This result suggests that Cut is dispensable for the onset of the DV boundary. This and the evolution predicted by modeling are in agreement with the in vivo results. In cut mutant discs, the early activation of Notch at the DV boundary, as shown by the expression of Wg, is comparable to wild-type discs. However, in mature third instar discs Notch activity and Wg expression are not maintained in the mutant background. Taken together, these results indicate that refractoriness of boundary cells to the Wg signal provided by the activity of Cut is required to shape a stationary and stable DV boundary in the developing wing primordium (Buceta, 2007).

This study analyzed the properties of the regulatory network for the establishment and maintenance of the DV organizer in the Drosophila wing imaginal disc. Evidence is provided that that a mathematical model can convert the initial DV asymmetric expression pattern of Notch ligands into the DV symmetric and mutually exclusive domains of active receptor and Notch ligands in boundary and non-boundary cells, respectively. To model the network 'circuitry', and test and verify the proposal, advantage was taken of a combination between in vivo and in silico experiments that has allowed checking of the analytical and predictive capacity of the modeling (Buceta, 2007).

The most striking finding of this research is that a novel property is required in the regulatory network for a robust and stable maintenance of the DV organizer: namely boundary cells must be refractory to the Wg signal. This property is conferred by the activity of Notch through its target gene cut. The role of Cut in repressing the Wg signaling pathway in boundary cells, and Wg in repressing Notch in non-boundary cells, generates two mutually exclusive domains of Notch and Wg activities, corresponding to boundary and non-boundary cells, respectively. Consequently, Notch ligands and receptors are expressed in two distinct non-overlapping cell populations. This helps to restrict the width of the boundary population to few (two-three) cells and contributes to polarizing ligand-receptor signaling towards the boundary and not against it, i.e., flanking ligands signal Notch towards the boundary but not against it since down-regulation of the Notch pathway in non-boundary cells inhibits the receptors' activity in those cells. In addition, light has been shed on several dynamical properties of the network, such as the refinement of Notch activity (Buceta, 2007).

At the time the role of Cut in the repression of Delta and Serrate expression was described, Cut and the concomitant restriction of ligand expression to non-boundary cells were postulated to be essential for the stability of the DV boundary. However, the other negative input of Wg into the Notch pathway through the activity of Dishevelled was not taken into account. In silico results have predicted that a general repression of the Wg pathway is required for stable activity of Notch at the DV boundary. In vivo results indicate that this repression takes place at the level or upstream of Armadillo. In order to be refractory to the inhibitory effect of Dishevelled on Notch, this repression should be taking place close to Dishevelled if not further upstream in the Wg signaling cascade (Buceta, 2007).

Finally, the conclusions are placed into a broader context. Boundary formation between adjacent rhombomeres in vertebrates relies on the same Wnt/Notch-dependent regulatory network. Therefore, it is speculated that boundary cells also need to be refractory to the Wnt signal to generate stable boundaries. To close, it is concluded that the robustness and stability of this network, in which the interconnectivity of the elements is crucial and even more important than the value of the parameters used, might explain its use in boundary formation in other multicellular organisms (Buceta, 2007).

Dynamic decapentaplegic signaling regulates patterning and adhesion in the Drosophila pupal retina; Rst activity opposes DE-cadherin-mediated cell adhesion

The correct organization of cells within an epithelium is essential for proper tissue and organ morphogenesis. The role of Decapentaplegic/Bone morphogenetic protein (Dpp/BMP) signaling in cellular morphogenesis during epithelial development is poorly understood. In this paper, the developing Drosophila pupal retina -- looking specifically at the reorganization of glial-like support cells that lie between the retinal ommatidia -- was used to better understand the role of Dpp signaling during epithelial patterning. The results indicate that Dpp pathway activity is tightly regulated across time in the pupal retina and that epithelial cells in this tissue require Dpp signaling to achieve their correct shape and position within the ommatidial hexagon. These results point to the Dpp pathway as a third component and functional link between two adhesion systems, Hibris-Roughest and DE-cadherin. A balanced interplay between these three systems is essential for epithelial patterning during morphogenesis of the pupal retina. Importantly, a similar functional connection has been identified between Dpp activity and DE-cadherin and Rho1 during cell fate determination in the wing, suggesting a broader link between Dpp function and junctional integrity during epithelial development (Cordero, 2007).

Loss of Dpp pathway activity results in a loss of epithelial integrity, but the function of Dpp signaling during maturation of developing epithelia is not fully understood. This study shows that reducing the activity of components of the Dpp pathway leads to abnormal Interommatidial precursor cells (IPC) shape and organization within the ommatidial hexagonal pattern. This activity is linked to fine regulation of apical junction components and is required to maintain stable cell-cell contacts during cell movements within the epithelium. The expression of Dpp in primary pigment cells and the segregation of its receptors to the neighboring IPCs suggest a model in which Dpp acts in the primaries to organize local IPCs through the dynamic control of apical junctions. This view is supported by the dynamic changes in p-Mad activity in the neighboring IPCs, which is highest during the stage (20-26 hours APF) when IPCs rearrangements are maximal (Cordero, 2007).

The role of Dpp in cellular morphogenesis during epithelial development is poorly understood. Therefore, advantage was taken of the unique stereotyped pattern of the pupal retina to study cell behavior as morphogenesis progresses, focusing on events at the single-cell level. In situ visualization experiments suggest that IPCs with reduced Tkv activity are incapable of maintaining their cell-cell contacts and are subject to aberrant changes in their cell shape. Further emphasizing the link with cellular adhesion, this function of Dpp signaling involves DE-cadherin and Rho1, which are essential regulators of cell adhesion and cell shape (Cordero, 2007).

Several lines of evidence are provided indicating that Rst is a negative regulator of Dpp signaling. Previous work has demonstrated that Rst directs IPC movements through selective cell adhesion: IPCs seek to maximize their Rst-mediated contacts with primaries while decreasing contacts with their neighbors. Additionally, reducing Rst activity leads to a failure of initial cell movement. Consistent with these results, Rst activity opposes DE-cadherin-mediated cell adhesion. One model to account for these observations is that cells require a balance between cell movement provided by Hibris-Rst and the stability of cell-cell contacts provided by Dpp signaling. Live visualization supports the view that reducing Dpp activity leaves cells with an imbalance, as IPCs move toward their proper positions but fail to stabilize cell-cell contacts or lock stably into their final positions. Furthermore, downregulation of Dpp signaling leads to unstable DE-cadherin IPC-IPC junctions. Conversely, loss of rst results in loss of cell movements, which can be compensated by either reducing cell adhesion or Dpp signaling activity, again supporting the importance of maintaining a balance between the Hbs-Rst and the Dpp-DE-cadherin systems. Perhaps Dpp (and, by extension, BMP) activity is utilized in the adult for similar functions -- for example, as a 'proof-reading' mechanism to remove aberrant cells from an epithelium (Cordero, 2007).

The results in the wing raise the interesting possibility that regulation of DE-cadherin and Rho1-dependent cell shape and cell adhesion might be a characteristic of Dpp pathway activity common to other biological systems. Similar to the pupal retina, epithelial cells in the wing disc with reduced Dpp signaling displayed abnormal morphologies and were unable to maintain their positions. In the case of the wing, these defects were manifested as viable cysts of mutant cells that were basally excluded from the epithelium. The mechanisms involved in such cell behaviors remain unknown. The results suggest that the role of Dpp signaling during wing patterning also involves DE-cadherin and Rho1. The experiments do not distinguish whether the defects in wing cell fates are a direct or a secondary effect of altered cell adhesion, although altering DE-cadherin activity by itself was not sufficient to cause such defects. Cell adhesion and cell fate have been related previously: for example, Rho-dependent cell shape changes can influence fate decisions in stem cells. Despite the commonalities observed, tissue-specific factors are likely to regulate Dpp-dependent epithelial patterning: for example, Rst does not appear to have a role in wing development, and no changes in retinal Tubulin distribution reported has been reported for the wing (Cordero, 2007).

Dpp is the closest ortholog of vertebrate BMP2/4, and it appears to be active during cellular morphogenesis in a number of contexts including the developing vertebrate eye. Interestingly, and similar to observations for IPCs, fiber cells in the developing vertebrate lens show high levels of p-SMAD activity during the period of cell elongation. Loss of the Type I receptor ALK3 (also known as BMPR1A) or expression of the inhibitor noggin led to abnormal morphogenesis of these fiber cells including mispositioning and failure to elongate; requirements for E-cadherin (also known as cadherin 1) and RHOA function have not been explored (Cordero, 2007).

Finally, Rst does regulate developmental processes other than IPC patterning. For example, Rst is expressed in retinal axons and is required for correct targeting of those axons into the larval brain lobes. Interestingly, Dpp signaling also has a role in this process. Genetic interactions between rst3 and members of the Dpp pathway in the arrangement of these descending axons, raising the intriguing possibility that the two systems act together in axon targeting as well (Cordero, 2007).

These results provide evidence to support a model in which the Dpp pathway acts as an intermediary between the Rst and DE-cadherin adhesion systems. A balanced interplay between these three systems is essential to regulate epithelial cell movements, cell shape and cell-cell contacts during morphogenesis of the pupal retina. Several questions emerge from this study. For example, the data suggest that Rst acts on Dpp signaling by regulating surface-associated Tkv. Immunoprecipitation experiments failed to identify a physical interaction between Rst and Tkv, suggesting intermediate steps remain to be identified. Also, the transcription factor Mad is required to regulate IPC patterning, but the transcriptional targets that link Dpp signaling to DE-cadherin and Rho1 are unknown. A better understanding of the links between these three pathways should help shed light on the mechanisms that regulate the fine cellular events required during patterning of developing epithelia (Cordero, 2007).

Polychaetoid controls patterning by modulating adhesion in the Drosophila pupal retina

Correct cellular patterning is central to tissue morphogenesis, but the role of epithelial junctions in this process is not well-understood. The Drosophila pupal eye provides a sensitive and accessible model for testing the role of junction-associated proteins in cells that undergo dynamic and coordinated movements during development. Mutations in polychaetoid (pyd), the Drosophila homologue of Zonula Occludens-1, are characterized by two phenotypes visible in the adult fly: increased sensory bristle number and the formation of a rough eye produced by poorly arranged ommatidia. It was found that Pyd is localized to the adherens junction in cells of the developing pupal retina. Reducing Pyd function in the pupal eye results in mis-patterning of the interommatidial cells and a failure to consistently switch cone cell contacts from an anterior-posterior to an equatorial-polar orientation. Levels of Roughest, DE-Cadherin and several other adherens junction-associated proteins are increased at the membrane when Pyd protein is reduced. Further, both over-expression and mutations in several junction-associated proteins greatly enhances the patterning defects caused by reduction of Pyd. These results suggest that Pyd modulates adherens junction strength and Roughest-mediated preferential cell adhesion (Seppa, 2008).

The data demonstrate that Pyd is an AJ-associated protein that is required for patterning of the pupal lattice cells. Live imaging of the developing eye indicates that Pyd is necessary for the directed movements of interommatidial precursor cells (IPCs) that allow cell sorting into defined niches. Membrane contacts are dynamically exchanged in the pupal eye: each shift in the position of a cell requires the removal of previous contacts and the establishment of new ones. Pyd regulates patterning at least in part through modulating levels of the AJ-associated proteins DE-Cadherin, β-Catenin, and α-Catenin. Other studies have suggested that cell adhesion is necessary both to facilitate and restrict cell movement within the eye epithelium; the interplay between these two processes requires tight regulation of the levels of both cell adhesion molecules and junctional proteins. The data indicate that removal of Pyd from the AJ compromises this tightly-regulated system and biases the cells toward poorly-directed movements, perhaps because of dysregulation of the timing or function of the mechanisms that control the stability of AJ proteins. This failure in precise regulation of adhesion was also highlighted in the inability of cone cells to exchange their membrane contacts: the apical interfaces of pyd-RNAi expressing cone cells were locked in place. Ectopic DE-Cadherin further increased the percentage of ommatidia affected, again emphasizing the link between pyd activity and the AJ (Seppa, 2008).

The localization of Pyd to the AJ in the pupal eye is dependent on both DE-Cadherin and α-Catenin. However, it was found that ectopic expression of either junctional protein is not sufficient to alter the localization of Pyd. Taken together, these data indicate that DE-Cadherin and α-Catenin are necessary to build or maintain the AJ and to localize Pyd but that, in excess, they are not sufficient to attract ectopic Pyd. This suggests that either Pyd protein levels are not easily altered or that Pyd may be binding to proteins other than the core AJ constituents. Recent work demonstrated that E-Cadherin was necessary for the initial steps of AJ formation while α-Catenin was essential for both the establishment and maintenance of the junction; only when α-Catenin was reduced was ZO-1 lost from established junctions. The results suggest that in dynamically restructured tissues such as the eye, both E-Cadherin and α-Catenin are necessary for the localization of AJ-associated proteins (Seppa, 2008).

The immunoglobulin superfamily member Roughest is necessary for appropriate sorting of IPCs during pupal eye development. Reducing Pyd increased Roughest protein levels specifically at the AJ. Roughest is the Drosophila orthologue of Neph1, a cell adhesion molecule necessary for the structure and function of the glomerular slit diaphragm in the mammalian kidney. The slit diaphragm is the main size-selective barrier in the filtration apparatus of the kidney and retains many characteristics of both the tight and AJ complexes from which it was derived. The Hibris orthologue Nephrin also forms part of the physical structure of the slit diaphragm and both cell adhesion molecules have been reported to bind to each other as well as to ZO-1. Perhaps ZO-1, as with Pyd, has a role in regulating the localization or levels of cell adhesion molecules such as Neph1 and Nephrin (Seppa, 2008).

The Dpp pathway has emerged as a major contributor to patterning of the Drosophila pupal eye. Its role requires functional connections to both DE-Cadherin and Roughest. For example, mutations in shotgun (the locus that encodes DE-Cadherin) suppresses the roughest eye phenotype but enhances Dpp pathway-dependent phenotypes in the eye and wing. Together, these data suggest a model in which (1) Roughest acts to promote the stability of membrane contacts to drive directed cell movements and (2) the Dpp pathway and Pyd act to destabilize the adherens junction complex and local cell contacts to allow for proper IPC sorting. Consistent with this view, it was observed that reducing pyd enhances the effects of reduced Dpp pathway activity in the eye and wing. Thus, Pyd appears to act in concert with the Dpp pathway to regulate select core components of the AJ during development (Seppa, 2008).

This study has shown that Pyd is required specifically for patterning the interommatidial cells of the Drosophila pupal eye. Pyd appears to regulate both cell shape and cell positioning by controlling the levels of AJ proteins such as DE-Cadherin and adhesion proteins such as Roughest. Thus, Pyd provides a link between adhesion and junction formation; a further understanding of its role in the pupal eye will shed light on how these processes are coordinated to generate precise cellular movements during epithelial patterning (Seppa, 2008).

Adult

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).

Wingless signaling initiates mitosis of primordial germ cells during development in Drosophila

The germline cells of Drosophila are derived from pole cells, which form at the posterior pole of the blastoderm and become primordial germ cells (PGCs). To elucidate the signal transduction pathways for the development of embryonic PGCs, the effects of various growth factors on the proliferation of PGCs were examined. Up- and down-regulation of Wingless (Wg) in both of soma and PGCs caused an increase and a decrease in the number of PGCs, respectively. The Wg/beta-catenin signaling pathway began to occur in PGCs at the same time as the PGCs began to divide during the embryonic stage in both sexes. In addition, PGCs were found to produce wg mRNA as they begin to divide. Thus, Wg functions as an autocrine factor to initiate mitosis in embryonic PGCs. Decapentaplegic affected the growth of PGCs from the end of the embryonic stage. The results indicate that these growth factors regulate the division of embryonic PGCs in a stage-specific manner (Sato, 2008).

The present study clearly shows that the Wg signal has an essential role in the initiation of mitosis in embryonic PGCs. Wg functions as an autocrine growth factor for the reentry of PGCs into mitosis. This is much different from the situation with regard to adult GSCs. The maintenance and division of GSCs have been shown to be regulated by niche cells that produce Dpp and Gbb. Wg regulates the maintenance of somatic stem cells but not GSCs. Wg had no effect on the growth of bam GSCs in culture (Niki, 2006). The simultaneous occurrence of the Wg signaling pathway and reentry of PGCs into mitosis in both sexes indicates that the sexual dimorphism of the mitotic property of PGCs during embryonic development would depend on the action of the Wg signaling pathway. At present, it is an open question what upstream factor(s) regulates the timing of Wg signaling in PGCs during embryonic development in each sex. One of the factors might be the JAK/STAT pathway, which was shown to be required for male-specific germ cell behavior during early embryonic development (Wawersik, 2005). When Upd was overexpressed in female embryos, extraordinary mitosis was induced at the mid-embryonic stage (Wawersik, 2005). It is important to elucidate the factor(s) that regulates the Wg signaling pathway for the initiation of the reentry into mitosis of embryonic PGCs in the two sexes (Sato, 2008).

Wnt is a family of secreted proteins that regulate several physiological and pathological processes in the development and maintenance of various tissues. In Drosophila, Wg signaling promotes cell cycle progression in the embryonic Malpighian tubules and in the early larval imaginal wing disc. In mammals, Wnt/β-catenin signaling regulates the maintenance of various stem cell systems, including intestinal epithelial, follicular, hematopoietic, and embryonic stem cells. Unexpectedly, the present results showing that the role of Wg signal in initiating the mitosis of embryonic PGCs are contrary to the role of Wnt signaling in PGC development in mouse. In mouse PGCs, nuclear-localized β-catenin gradually disappears after E13.5 (Kimura, 2006). The suppression of Wnt/β-catenin signaling is a prerequisite for normal cell cycle progression in PGCs. The cell cycle is arrested by overexpression of β-catenin, resulting in germ cell deficiency followed by apoptosis. At present, the reason for Wg to have different roles in PGCs of fly and mouse are not known. Further work will be required to elucidate the function of Wg in PGCs of diverse animals (Sato, 2008).

Considering that in the present study the percentage of mitotic PGCs reached the same level in the later embryonic stage even when Wg was down-regulated constitutively with a tissue-specific driver, the lack of Wg signaling does not affect the function of Dpp, which becomes effective from the late embryonic stage. During larval development, only Dpp is effective in promoting the growth of PGCs (Sato, unpublished results). Dpp may function as a main growth factor for the division of embryonic larval development. Further analyses of the growth factors that regulate the division of PGCs after the embryonic stages are needed (Sato, 2008).

The increase in the number of PGCs was always less than 2-fold compared with controls. This may be due to a limitation in the time schedule and space available for gonads during development. Recently, Gilboa (2006) showed that the proliferation of PGCs and survival of the intermingled somatic cells that contacted them were coordinated by means of a feedback mechanism composed of a positive signal and a negative signal during gonad development. They proposed that homeostasis and coordination of growth between soma and germ line in the larval ovary were achieved using a sensor of PGC numbers (epidermal growth factor-mediated survival of intermingled cells) coupled to a correction mechanism inhibiting PGC proliferation. Thus, it is reasonable to consider that the number of PGCs is controlled by this mechanism (Sato, 2008).

Drosophila apc regulates delamination of invasive epithelial clusters

Border Cells in the Drosophila ovaries are a useful genetic model for understanding the molecular events underlying epithelial cell motility. During stage 9 of egg chamber development they detach from neighboring stretched cells and migrate between the nurse cells to reach the oocyte. RNAi screening led to the identification of the dapc1 gene as being critical in this process. Clonal and live analysis showed a requirement of dapc1 in both outer border (oBC) cells and contacting stretched cells (SCs) for delamination. This mutant phenotype was rescued by dapc1 or dapc2 expression. Loss of dapc1 function was associated with an abnormal lasting accumulation of β-catenin/Armadillo and E-cadherin at the boundary between migrating border and stretched cells. Moreover, β-catenin/armadillo or E-cadherin downregulation rescued the dapc1 loss of function phenotype. Altogether these results indicate that Drosophila Apc1 is required for dynamic remodeling of β-catenin/Armadillo and E-cadherin adhesive complexes between outer border cells and stretched cells regulating proper delamination and invasion of migrating epithelial clusters (De Graeve, 2012).

Cell migration is a dynamic process involving multiple cell-cell and cell-substrate interactions. It is therefore important to better characterize the molecular mechanisms underlying cell adhesion during all stages of cell invasion. BC migration represents a powerful in vivo model, as cells become motile through a multi-step process involving cluster assembly and cohesion, delamination from the follicular epithelium, and labile interactions with nurse cells throughout migration. All these processes require the dynamic remodeling of DE-cadherin and β-catenin/Arm during adhesion. Indeed, an artificial DE-cadherin-β-catenin/Arm fusion protein can act as a strong dominant negative preventing BC migration (De Graeve, 2012).

Several mechanisms can regulate DE-cadherin - β-catenin/Arm interactions. In cell culture systems, Apc proteins have been shown to be able to compete with E-cadherin for β-catenin/Arm binding. As a result, β-catenin/Arm is continuously incorporated into and released from adherens junctions. Hence β-catenin/Arm exchange is strongly affected in cells containing mutations in the apc gene. Nevertheless, little is known about the molecular events involved in cell delamination and about the role of Apc in this process. In order to better understand how cell-cell and cell-substrate interactions control BC migration, an RNAi-based genetic screen was performed and dapc1 was identified as a key regulator of BC delamination (De Graeve, 2012).

The results show that dApc1 regulates BC delamination through DE-cadherin - β-catenin/Arm remodeling at the interface between oBC and SC. Indeed, loss of dapc1 function in oBC and adjacent SC led to abnormal persistence of DE-cadherin and β-catenin/Arm proteins at their boundary preventing them to detach from neighboring cells. Down regulation of β-catenin/arm or DE-cadherin rescued the dapc1 mutant phenotype indicating that loss of dapc1 function indeed increases adhesion strength between oBC and adjacent SC. The results fit with a model in which dApc1 regulates BC-SC adhesion acting at two levels. First, dApc1 competes with DE-cadherin for β-catenin/Arm binding, hence regulating the interaction between DE-cadherin and β-catenin/Arm and thereby adhesion remodeling. Second, dApc1 also favors β-catenin/Arm degradation, thereby controlling the level of proteins involved in adhesion. Altogether, this allows dApc1 to regulate negatively global adhesion strength in between oBC and SC and control BC delamination (De Graeve, 2012).

In the absence of dApc1, the half-life of β-catenin/Arm is sustained, its interaction with DE-cadherin is favored and as a consequence cells display at their surface a higher number of stable adhesions. The dapc1 over-adhesive phenotype can be rescued by lowering β-catenin/Arm levels, rendering delamination again possible. Surprisingly, overexpression of β-catenin/Arm in dapc1 mutant cells also generated a partial rescue of the dapc1 mutant phenotype. Although most of the BC clusters did not reach the oocyte in time, some mutant clusters were able to delaminate. Overexpression of β-catenin/Arm probably bypasses the need of dApc. Indeed excess of β-catenin/Arm molecules generates inter-molecular competition for DE-cadherin binding, rescuing partially BC delamination. In contrast to wild type β-catenin/Arm, the overexpression of ArmS10 did not rescue the dapc mutant phenotype. ArmS10 lacks a sequence (aa 34-87) that contains a consensus GSK-3β phosphorylation site leading to the degradation of wild type β-catenin/Arm protein after phosphorylation. This suggests that β-catenin/Arm phosphorylation by GSK3-β kinase is required for BC delamination. The results are consistent with previous data in mammals showing the presence of Apc and β-catenin/Arm containing complexes that are phosphorylated by GSK3β/CKI, favoring their degradation (De Graeve, 2012).

The dapc mutant phenotype requires loss of dapc both in oBC and adjacent SC. Indeed, when mutant BCs interact with wild type SCs, or vice-versa, abnormal accumulation of β-catenin/Arm and DE-cadherin is no longer detectable in between the mutant and wild type cells and BC delaminate, migrate and reach the oocyte normally. This suggests that in the absence of dapc, DE-cadherin from the mutant cell can still establish a functional interaction with DE-cadherin from the wild type cell. The remodeling of β-catenin/Arm-DE-cadherin in the wild type cell is probably sufficient to allow the release of the mutant cell (De Graeve, 2012).

Live imaging of migrating BC clusters revealed that oBC change their position within the cluster throughout migration. Loss of dapc function led to persistent β-catenin/Arm and DE-cadherin at cell boundaries (oBC-oBC and oBC-SC interfaces), therefore potentially increasing cluster stiffness. However, no defect was observed in mutant oBC tumbling or cluster velocity, suggesting that dApc1 is not essential for regulating inter-oBCs interactions during migration (De Graeve, 2012).

Several models from cell culture to mouse have been used to study Apc function. Wild type Apc acts as a scaffold for many proteins including F-actin, microtubules, β-catenin/Arm, and regulates multiple biological processes independent of Wg signaling, such as chromosomal segregation, cell adhesion, cell migration and apical cell extrusion. The current results show that BC migration provides a new powerful model, out of Wg influence, unraveling mechanisms regulating collective cell migration in vivo with important implications for wound healing and tumor metastasis (De Graeve, 2012).

Effects of Mutation or Deletion

The phenotype and molecular lesions generated by different arm mutations have been compared. Severely truncated proteins retain some function; the degree of function is strictly correlated with the length of the truncated protein, suggesting that the internally repetitive ARM protein is modular in function (Peifer 1990).

Analysis of double mutants demonstrates that Armadillo's role in wingless signaling is direct, and that Armadillo functions downstream of both wingless and zeste-white 3 (Peifer, 1994a).

In embryos mutant for armadillo, dishevelled and porcupine, the changes in engrailed expression are identical to those in wingless mutant embryos, suggesting that their gene products act in the wingless pathway (van den Heuvel (1993). dsh and porc act upstream of zw3, and arm acts downstream of zw3 (Siegfried, 1994).

In embryos mutant for hedgehog, fused, cubitus interruptus and gooseberry, expression of engrailed is affected to varying degrees. However wingless expression in the latter group decays in a similar way earlier than engrailed expression, indicating that these gene products might function in the maintenance of wingless expression (van den Heuvel, 1993).

Programmed cell death plays an essential role in the normal embryonic development of Drosophila. One region of the embryo where cell death occurs, but has not been studied in detail, is the abdominal epidermis. Because cell death is a fleeting process, time-lapse, fluorescence microscopy was used to map epidermal apoptosis throughout embryonic development. Cell death occurs in a stereotypically striped pattern near both sides of the segment border and to a lesser extent in the middle of the segment. Approximately three-quarters of the dying cells appear in or immediately adjacent to the en stripe. The rest of the apoptotic nuclei are located in the middle of the segment. It appears that two rows of cells on either side of the segmental border die in each segment of the ventral ectoderm during stages 12-14. There is also an apparent clustering of apoptotic nuclei at specific locations along the dorsal-ventral axis. The number of cell deaths occurring within the 2-3 cells either side of the segment boundary was counted and this was compared with 5-6 cells in the middle of the segment. Considering that a segment is 10-12 cells across, this accounting partitions the segment into two equal-sized groups: the segment border cells and mid-segment cells. The segment border cells includes the en cells plus one or two rows of cells anterior and two or three cell rows posterior to the en stripe. Analysis of four time-lapse recordings of wild-type embryos, where eight segments were scored per embryo, shows that 73% of cell death occurs in the segment border cells and the remaining 27% occurs in the mid-segment cell (Pazdera, 1998).

This map of wild-type cell death was used to determine how cell death patterns change in response to genetic perturbations that affect epidermal patterning. Previous studies have suggested that segment polarity mutant phenotypes are partially the result of increased cell death. Mutations in wingless, armadillo, and gooseberry lead to dramatic increases in apoptosis in the anterior of the segment while a naked mutation results in a dramatic increase in the death of engrailed cells in the posterior of the segment. When wg function is disrupted during stage 11 (the fate specification phase of epidermal development) approximately two rows of cells die in the anterior-most portion of each segment during stages 12-14. These dying cells are approximately 6 rows of cells away from the Wg-secreting cells. An arm mutation that disruptes Wg signaling also eliminates the same rows of cells. These results show that Wg signaling is required at a distance to promote the survival of cells in the anterior of the segment. It is important to note that the cells expressing Wg prior to the temperature shift and those cells immediately anterior to Wg-secreting cells do not die and, therefore, may be more resistant to apoptosis than those cells at a distance. Mutations in the segment polarity genes gsb also lead to the death of cells in the anterior of the segment. However this death is more restricted to the ventral surface (Pazdera, 1998).

Strong mutations in nkd results in a cuticle phenotype opposite that of wg mutations. Previous genetic studies have suggested that the nkd gene product is necessary to suppress the domain of Wg function. nkd mutants also evince increased cell death, of which the majority is located in the expanded Engrailed-expressing domain. There is however no increase in cell death in the anterior of the segment similar to that seen in Wg mutants. Taken together these results suggest that two separate systems may be involved in promoting cell survival in the embryonic ectoderm: a nkd-dependent system that keeps posterior cells alive and a wg-dependent system that plays a similar role in the anterior of the segment. These two systems may not be mutually exclusive. It has been suggested that cell death in a wg mutant is suppressed by a nkd mutation (Bejsovec, 1993). These results provide evidence that segment polarity gene interactions play an intimate role in epidermal cell survival. However, much more work is needed to further understanding of these processes (Pazdera, 1998).

An intermediate mutant allele of armadillo was used to explore the requirement for ARM in adherens junction assembly, cell polarity and morphogenesis in Drosophila. Adherens junctions cannot assemble in the absence of ARM; this leads to dramatic defects in cell-cell adhesion. The epithelial cells of the embryo lose adhesion to one another, round up, and apparently become mesenchymal. In arm mutants, alpha-catenin no longer accumulates at the plasma membrane, but instead is found diffusely in the cytoplasm. Shotgun, the Drosophila E-cadherin, is normally tightly localized to the plasma membrane and enriched in adherens junctions, but in arm mutants, Shotgun accumulation at the plasma membrane is reduced. Much of the remaining Shotgun accumulates within cells, presumably in the ER, Golgi, or endosomes. This may be a result of endocytosis. Mutant cells also lose their normal cell polarity. These disruptions to the integrity of the epithelia constitute a block to the appropriate morphogenetic movements of gastrulation. There is little or no germ band extention, and the ventral furrow and posterior midgut fail to invaginate normally. Crumbs protein does not appear to be required for initial assembly of adherens junctions, or for early cell polarity. Genetic interactions between armadillo and crumbs are additive, with no dosage sensitive interactions, suggesting that they may not be required together early in development. In contrast, reducing Shotgun levels suppresses the armadillo segment polarity phenotype. It has been suggested that this suppression reflects the fact that Armadillo's roles in adherens junctions and Wingless signaling are separable, and that under conditions where ARM is limiting, the reduction in the number of junctional complexes frees up some of the wild-type ARM, allowing it to function in the Wingless signaling. Armadillo is also required in oogenesis. The earliest defect seen in arm null mutant egg chambers is failure in adhesion between follicle and germ cells. Centripetal follicle cells frequently fail to migrate and separate the oocyte from the nurse cells, while nurse cells often fail to transfer their contents into the oocyte (Cox, 1996).

The zonula adherens (ZA) belongs to a family of actin-associated cell junctions called adherens junctions. Antibodies specific to cellular junctions and nascent plasma membranes have been used to study the formation of the zonula adherens in relation to the establishment of basolateral membrane polarity. The same approach was then used as a test system to identify X-linked zygotically active genes required for ZA formation. ZA formation begins during cellularization; the basolateral membrane domain is established at mid-gastrulation. By creating deficiencies for defined regions of the X chromosome, genes have been identified that are required for the formation of the ZA and the generation of basolateral membrane polarity. Embryos mutant for both stardust (sdt) and bazooka (baz) fail to form a ZA. In addition to the failure to establish the ZA, the formation of the monolayered epithelium is disrupted after cellularization, resulting by mid-gastrulation in formation of a multilayered cell sheet. Electron microscope analysis of mutant embryos reveals a conversion of cells exhibiting epithelial characteristics into cells exhibiting mesenchymal characteristics. To investigate how mutations that affect an integral component of the ZA itself influences ZA formation, embryos with reduced maternal and zygotic supply of wild-type Arm protein were studied. These embryos, like embryos mutant for both sdt and baz, exhibit an early disruption of ZA formation. These results suggest that early stages in the assembly of the ZA are critical for the stability of the polarized blastoderm epithelium (Müller, 1996).

A screening was carried out to identify components of the wingless signal transduction pathway; the screen sought dominant suppressors of the rough eye phenotype, caused by a transgene that drives ectopic expression of wingless during eye development. Essentially such a screen would identify cellular components that when mutated would fail to transduce signals from ectopically produced Wingless. Four strong suppressors were found that fall into two complementation groups. Two are recessive alleles of armadillo and two are recessive alleles of a locus on the fourth chromosome, designated as pangolin. A stronger allele of pan was isolated which shows strong genetic interactions when trans-heterozygous with either of the two isolated armadillo mutations, causing adult phenotypes characteristic of reduced wg activity. These results suggest that pan, like arm, encodes a component of the wingless transduction pathway, and also raise the possiblity that these components may physically interact (Brunner, 1997).

The Drosophila retina is made from hundreds of asymmetric subunit ommatidia arranged in a crystalline-like array, with each unit shaped and oriented in a precise way. One explanation for the precise cellular arrangements and orientations of the ommatidia is that they respond to two axes of polarized information present in the plane of the retinal epithelium. Earlier work has shown that one of these axes lies in the anterior/posterior(A/P) direction and that the polarizing influence is closely associated with the sweep of the Hedgehog-dependent morphogenetic wave (see Progression of the morphogenetic furrow across the eye disc). Evidence is presented for a second and orthogonal axis of polarity: this signal can be functionally separated from the A/P axis. The polarizing information acting in this equatorial/polar axis (Eq/Pl) is established in at least two steps -- the activity of one signaling molecule functions to establish the graded activity of a second signal. Ectopic Wg expression results in two significant effects. (1) Clones are generated with associated polarity inversions. (2) Although significant changes in retinal polarity are associated with the clones, the distance over which the effect is exerted is restricted to from between 7 to 2 ommatidial rows. Ectopic Wg clones have two distinct features with respect to their polarity effects: (1) the aberrant polarity is asymmetrically distributed in relation to the clone (greater changes in polarity occur in polar positions relative to the center of the clone), and (2) the potency of the Wg-expressing clones to induce polarity reversals show maximial polarity-reversal effects at the equator and minimal effects at the pole (Wehrli, 1998).

Other genes downstream of wingless also appear associated with eye Eq/Pl polarity. The product of the arrow (arr) gene has been placed in the Wingless pathway based on a number of criteria:

To a variable extent, clones of armadillo and dishevelled induce polarity inversions on their equatorial side. The critical observation is that mutations in these recognized transducers of the Wg signal induce non-autonomus effects, consistent with their regulating the activity of a sendary signaling factor. This secondary signal is termed factor-X. Not only do arr, arm and dsh clones specifically affect the equatorial side, they are also more potent in achieving this at the pole than the equator. Thus it is inferred that factor-X activity is graded in the Eq/Pl axis but there is insufficient information to determine whether the activity is high at the equator and low at the poles, or vice-versa (Wehrli, 1998).

Third instar larval eye imaginal discs (the precursors of the adult eye) from homozygous APC-like mutants were analyzed prior to development of the photoreceptor phenotype, to determine whether photoreceptor cell loss is a consequence of defects in the initial formation of neurons or from defects in their subsequent differentiation. The mutant eye discs were examined for expression of Neurotactin, a neuronal-specific transmembrane protein that is used as a marker for photoreceptor cells. Both at the level of patterning of the ommatidial arrays and at the level of individual photoreceptor cells, the Neurotactin antigen staining is normal in the Apc mutant. As well, retinal axonal projections to the optic lobe are intact. This indicates that the initial photoreceptor cell formation proceeds normally in Apc mutants and that the defect seen in the mutant adult is the result of neuronal degeneration. Further studies have determined that the neuronal degeneration in the Apc mutant is a result of programmed cell death (apoptosis). Expression of p35, a baculoviral protein that interferes with apoptosis by inhibiting the function of caspase proteases, rescues the Apc mutant phenotype. Despite the dramatic rescue from death of Apc mutant retinal neurons by p35, there is a striking feature that distinguishes the rescued photoreceptors. In wild-type adult eyes, the retinal neurons extend the entire length of the ommatidia, tapering gradually in diameter from their apical to basal regions. In contrast, while the rescued Apc mutant photoreceptors display an intact morphology at the apical surface of the eye, at more basal levels, their diameters are dramatically shrunken, and they lose contact with their neighbors. This abnormal morphology is thought to reflect an arrest in differentiation and that this arrest accompanies the apoptotic death observed in the Apc mutant eye (Ahmed, 1998).

To test for Apc regulation of Arm activity genetically, Arm levels in the Apc mutant were lowered by replacing one wild-type copy of the arm gene with an null allele. When the wild-type arm gene dosage is reduced by one-half in the Apc mutant, many neurons survive. The overall efficiency of rescue achieved by halving the Arm dosage is similar to that obtained by ectopic p35 expression; however, one striking difference between the two is that the neurons rescued in the arm heterozygotes appear completely normal from apex to base. Thus, an inactivating arm mutation is a dominant suppressor of both the differentiation defect and the cell death of retinal neurons in the Apc mutant. These findings provide genetic evidence that Apc functions to regulate Arm negatively and suggest that in the absence of Apc, an increase in Arm activity results in both a differentiation defect and apoptotic cell death (Ahmed, 1998).

To test this idea, the effects of elevated levels of Arm on photoreceptor differentiation were analyzed using the UAS/GAL4 system to overexpress Arm in retinal neurons. Neuronal-specific overexpression of Arm under control of the elav-GAL4 transactivator results in photoreceptor loss that is phenotypically similar to, but weaker than, that seen in Apc mutants, with some ommatidia losing all photoreceptor cells, and most others having a reduction in their number. This reveals that death of photoreceptors is sensitive to Arm dosage and suggests the requirement for titration of Apc activity. To examine further the effects of Arm levels on photoreceptor death, mutations in the amino terminus of Arm were used that reduce the rate at which it is degraded. Neuronal-specific overexpression of an amino-terminal deletion of Arm that results in its stabilization, directed by the elav-GAL4 transactivator, results in the loss of all neurons in all ommatidia, and only pigment cells remain. Overexpression of stabilized Arm under control of the sevenless-GAL4 transactivator, which directs strong expression in 3 of the 8 photoreceptor cells, and weak expression in 2 others, results in photoreceptor loss of only a fraction of cells per ommatidium. Together, these findings demonstrate that overexpression of Arm within retinal cells committed to a neuronal fate results in their death and suggest that the Apc mutant phenotype is mediated by elevation of Arm activity (Ahmed, 1998).

Since Arm is a multifunctional protein, specific mutants of arm that allow a dissection of its distinct functions were examined in a Apc mutant background to delineate regions that are required in the induction of cell death. The armH8.6 mutant allele creates a truncation of Arm's carboxyl terminus that reduces Arm's ability to mediate Wingless signaling in vivo. Flies heterozygous for the armH8.6 allele were examined to determine whether deletion of the carboxyl terminus reduces Arm's ability to induce apoptosis in a Apc mutant background. Reduction of the wild-type gene dosage of arm by one-half due to the introduction of the null allele armYD35 rescues many photoreceptors from death and thereby dominantly suppresses the Apc mutant phenotype. In contrast, the mutant allele armH8.6 acts like a wild-type copy of the gene; all photoreceptors in all ommatidia degenerate completely. This unexpected finding suggests that the carboxyl terminus of Arm is not required for Arm's ability to induce photoreceptor death in the Apc mutant. A similar assay was utilized to analyze the requirement of other regions of Arm for the induction of cell death. A series of transgenes containing deletions within Arm have differential effects on Arm's roles in cell adhesion and Wingless signal transduction. These transgenes were expressed under control of the Arm promoter, and protein levels of these mutated forms of Arm were shown to be equivalent to wild-type Arm levels (Orsulic, 1996). Flies heterozygous for these mutated arm genes were examined to determine whether they retain the ability to induce apoptosis in a Apc mutant background. A series of deletions in Arm, termed S5, S15, and S12, eliminate Armadillo repeats 5, 8, and portions of 10 and 11, respectively. Each deletion severely disrupts Arm's ability to induce cell death in the Apc mutant. In contrast, a deletion in which Arm's alpha-catenin binding site is eliminated retains the ability to induce cell death. This finding is consistent with the results obtained by overexpression of stabilized Arm. Although this stabilized mutant Arm protein lacks amino acids required for binding to alpha-catenin, it retains the ability to induce photoreceptor death. Together, these findings suggest that while the carboxyl terminus and alpha-catenin binding site are dispensable for Arm-induced cell death, Arm's central repeats are required for this activity (Ahmed, 1998).

When a constitutively active from of armadillo is expressed in pangolin mutants, its action is largely inhibited. Such double mutants resemble pangolin mutants in that they have alternating denticles and naked cuticle, with portions of the lateral naked cuticle converted to denticles. However, they often have regions of naked cuticle intruding into the normal denticle belt at the ventral midline (van de Wetering, 1997).


armadillo: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | Effects of Mutation | References

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