Effects of Mutation or Deletion (part 2/2)

Armadillo Function in Oogenesis

The anterior-posterior axis of Drosophila oocyte originates early in oogenesis, when one of two pro-oocytes becomes selected to become the oocyte. This process occurs well before Gurken signaling as one of the earliest steps after the formation of the 16 cell cyst originating from a single cystoblast. The anterior-posterior axis originates from two symmetry-breaking steps during early oogenesis. First, one of the two pro-oocytes within the cyst of 16 germline cells is selected to become the oocyte. This cell then comes to lie posterior to the other germline cells of the cyst, thereby defining the polarity of the axis. The oocyte reaches the posterior of the cyst in two steps: (1) the cyst flattens as it enters region 2b of the germarium to place the two pro-oocytes in the center of the cyst, where they contact the posterior follicle cells; (2) one cell is then selected to become the oocyte and protrudes into the posterior follicle cell layer when the cyst rounds up on entering region 3. During this germ cell rearrangement, the components of the homophilic cadherin adhesion complex, DE-cadherin, Armadillo and alpha-catenin, accumulate along the border between the oocyte and the posterior follicle cells. The positioning of the oocyte requires cadherin-dependent adhesion between these two cell types, since the oocyte is frequently misplaced when DE-cadherin, known as Shotgun, is removed from either the germline or the posterior follicle cells. It is concluded that the oocyte reaches the posterior of the germline cyst because it adheres more strongly to the posterior follicle cells than its neighbours during the germ cell rearrangement that occurs as the cyst moves into region 3. The Drosophila anterior-posterior axis therefore becomes polarized by an unusual cadherin-mediated adhesion between a germ cell and mesodermal follicle cells (Gonzalez-Reyes, 1998).

The phenotypes produced by armadillo and shotgun mutant germline clones have suggested that Shotgun-mediated adhesion may be required either to localize the oocyte to the posterior of the cyst or to maintain it in this position as the egg chamber grows. To distinguish between these possibilities, germline clones that were mutant for four shotgun alleles of increasing strength were generated. All four mutants give rise to egg chambers with misplaced oocytes, and the penetrance of this phenotype correlates with the severity of the mutant allele. This phenotype is already apparent in the germarium. For example, although the region 2b cysts usually have a wild-type arrangement of germ cells in shg IH germline clones, the oocyte never protrudes into the follicle cell layer when the cyst enters region 3, and frequently occupies a lateral position. An identical phenotype is also seen in germline clones of a strong armadillo allele. Thus, Shotgun and Armadillo are required in the germ cells for the initial positioning of the oocyte at the posterior of the cyst during the cell rearrangement that takes place as the cyst moves into region 3. In addition to producing a very high frequency of misplaced oocytes in region 3, germline clones of shg IG29 disrupt the organization of the germ cells earlier in the germarium. The mutant cysts do not flatten in region 2b to form the 1-cell-thick disc that extends across the width of the germarium, and remain the same shape as region 2a cysts. shotgun mutants do not affect oocyte determination. Most known mutants that affect oocyte positioning seem to do so indirectly by disrupting the determination and differentiation of the oocyte. However, this does not appear to be case for shotgun and armadillo mutants, since all of the markers for oocyte differentiation examined are expressed normally in the misplaced oocytes produced by germline clones (Gonzalez-Reyes, 1998).

The cadherin family of adhesion molecules generally mediate homophilic adhesion between cells of the same type, and this is also the case for the Shotgun-dependent adhesion between the germ cells during the flattening of the cyst in region 2b, the first of two steps in which Shotgun is involved in the correct placement of the oocyte. The differential adhesion between the oocyte and the posterior follicle cells that occurs at the next stage is quite different, however, because these two cell types are completely unrelated and arise from separate lineages that are set aside at the earliest stages of embryogenesis. The oocyte is descended from the pole cells, which are the primordial germ cells that form at the posterior of the embryo about one and a half hours after fertilization, whereas the follicle cells arise from the gonadal mesoderm. This role for cadherin in heterotypic adhesion is very unusual, but not entirely without precedent. In mammals, E-cadherin has been shown to mediate adhesion between Langerhans cells and keratinocytes, while N-cadherin contributes to the attachment between developing spermatocytes and the Sertoli cells of the testis. It is interesting to note that the latter example also involves adhesion between germline and somatic cells (Gonzalez-Reyes, 1998 and references).

Shotgun (Shg) is an epithelial cadherin in Drosophila, and forms adherens junctions by associating with Armadillo (beta-catenin). To investigate its role in oogenesis, germ-line clones homozygous for a null mutation in shotgut were generated, and their phenotypes examined and compared with those of armadillo (arm) mutants. In the wild-type ovaries, Shotgun is expressed by both the germ-line and somatic derivatives, colocalizing with Armadillo. In shg mutant ovaries, when the mutation is restricted to the germ line, germ cells are rounded, and generate gaps between themselves, suggesting that their surface adhesiveness is either reduced or lost. However, the positioning of germ cells in the egg chamber is normal. Two groups of somatic follicle cells -- the border cells and centripetal follicle cells -- frequently migrate along incorrect pathways, indicating that DE-cadherin is required for their appropriate migration. Notably, the shg phenotypes are distinct from those of arm null mutants. Intercellular adhesion appears to be less severely affected by arm than by the shg mutation, and the actin-based cytoskeleton and cell arrangement are disorganized only in the arm mutants. These findings suggest that Shotgun is critical for cell-cell adhesion, and functional to a certain extent without Armadillo, whereas Armadillo is required for cytoskeletal organization and for the control of cell positioning. It is therefore proposed that the molecular complex of Shotgun and Armadillo that is present in normal cells is endowed with multiple functions derived from each molecule (Oda, 1997).

Identifying the signals involved in maintaining stem cells is critical to understanding stem cell biology and to using stem cells in future regenerative medicine. In the Drosophila ovary, Hedgehog is the only known signal for maintaining somatic stem cells (SSCs). Wingless (Wg) signaling is also essential for SSC maintenance in the Drosophila ovary. Wg is expressed in terminal filament and cap cells, a few cells away from SSCs. Downregulation of Wg signaling in SSCs through removal of positive regulators of Wg signaling, dishevelled and armadillo, results in rapid SSC loss. Constitutive Wg signaling in SSCs through the removal of its negative regulators, Axin and shaggy, also causes SSC loss. Also, constitutive wg signaling causes over-proliferation and abnormal differentiation of somatic follicle cells. This work demonstrates that wg signaling regulates SSC maintenance and that its constitutive signaling influences follicle cell proliferation and differentiation. In mammals, constitutive ß-catenin causes over-proliferation and abnormal differentiation of skin cells, resulting in skin cancer formation. Possibly, mechanisms regulating proliferation and differentiation of epithelial cells, including epithelial stem cells, are conserved from Drosophila to man (Song, 2003).

Wg produced from terminal filament and cap cells may reach SSCs at a distance of a few cells by either diffusion or active transport, and then Wg directly controls SSC maintenance. Furthermore, correct intermediate levels of wg signaling seem to be important for maintaining SSCs in the Drosophila ovary. Reduction of wg signaling in SSCs by removal of positive regulators such as arm and dsh causes rapid SSC loss, as does constitutive wg signaling in SSCs by removal of negative regulators such as Axn and sgg. wg signaling maintains SSCs through several possible mechanisms: (1) wg signaling could be required for SSC self-renewal and/or survival; (2) it could maintain the association of SSCs with IGS cells, and/or (3) both mechanisms could work simultaneously. DE-cadherin-mediated cell adhesion has been shown to be important for keeping SSCs in their niche; it also shares arm as a common component with wg signaling. wg signaling is known to regulate levels of arm, which are also important for DE-cadherin-mediated cell adhesion. Thus, it is possible that wg signaling regulates cell adhesion between SSCs and their niches. In addition, arm mutant clonal analysis strongly argues that wg signaling must also directly regulate SSC self-renewal and/or survival. arm2 mutant SSC clones are lost very quickly over time in comparison with wild-type SSC clones, and the arm2 mutation primarily affects wg signaling but does not disrupt DE-cadherin-mediated cell adhesion. Therefore, wg signaling controls SSC maintenance through regulating SSC self-renewal/survival and/or cell adhesion between SSCs and their niche cells. The temperature-sensitive allele of wg gives very mild phenotypes in follicle cell production, however, removal of wg downstream components has a dramatic impact on SSC maintenance. In Drosophila, there are six other wg-related genes. This raises an interesting possibility that other wg-like molecules could also be involved in regulating SSC maintenance (Song, 2003).

Armadillo and the Notch - Wingless Interaction

The development and patterning of the wing in Drosophila relies on a sequence of cell interactions molecularly driven by a number of ligands and receptors. Genetic analysis indicates that a receptor encoded by the Notch gene and a signal encoded by the wingless gene play a number of interdependent roles in this process and display very strong functional interactions. At certain times and places, during wing development, the expression of wingless requires Notch activity and that of its ligands Delta and Serrate. This has led to the proposal that all the interactions between Notch and wingless can be understood in terms of this regulatory relationship. This proposal has been tested by analyzing interactions between Delta- and Serrate-activated Notch signaling and Wingless signaling during wing development and patterning. Cell death caused by expressing dominant negative Notch molecules during wing development cannot be rescued by coexpressing Nintra. This suggests that the dominant negative Notch molecules cannot only disrupt Delta and Serrate signaling but can also disrupt signaling through another pathway. One possibility is the Wingless signaling pathway, since the cell death caused by expressing dominant negative Notch molecules can be rescued by activating Wingless signaling. Furthermore, the outcome of the interactions between Notch and Wingless signaling differs when Wingless signaling is activated by expressing either Wingless itself or an activated form of the Armadillo. For example, the effect of expressing the activated form of Armadillo with a dominant negative Notch on the patterning of sense organ precursors in the wing resembles the effects of expressing Wingless alone. This result suggests that signaling activated by Wingless leads to two effects: a reduction of Notch signaling and an activation of Armadillo (Brennan, 1999).

Expression of a dominant negative Notch molecule (Extracellular Notch or ECN) throughout the developing wing mimics the effects of loss of Notch function. However, Nintra cannot rescue the cell death caused by overexpressing ECN. Since Nintra provides constitutive signaling for Delta and Serrate during wing development and the effects of ECN are mediated by the sequestration of extracellular molecules that can interact with Notch, this suggests that the ECN molecule is sequestering extracellular molecules other than Delta and Serrate and attenuating signaling through another pathway. One candidate pathway is the Wingless signaling pathway, since the cell death caused by expressing the ECN can be rescued by activating Wingless signaling. Therefore, it is possible that the ECN molecule is sequestering the Wingless protein. The possibility that Wingless can bind the extracellular domain of Notch is supported by the results that are presented here, in particular, by two observations: first, that some of the deleterious effects of ECN can be suppressed by Wingless, but not Wingless signaling in the form of a constitutively active Armadillo molecule; and second, that this interaction requires specific EGF-like repeats of Notch, namely repeats 17-19 and 24-26 but not 10-12. Evidence for a physical interaction between Notch and Wingless has also been provided recently by Wesley (1999) who finds that the Wingless protein is enriched in a biopanning assay designed to identify proteins that interact with the extracellular domain of the Notch protein and that Wingless can be immunoprecipitated with Notch from embryo extracts and cultured cells. These experiments also show that the association of Wingless with Notch requires the integrity of a region of Notch centered around EGF-like repeats 24-26 (Wesley, 1999) which these experiments indicate are essential for the interactions that are described between Wingless and ECN during wing development and patterning (Brennan, 1999).

High levels of Wingless throughout the developing wing induce widespread development of sensory organs, an observation that correlates with the requirement for Wingless in this process during normal development. However, it is consistently observed that an activated form of Armadillo has a much weaker effect than Wingless on neural development. However, the difference is unlikely to be due to a weak UASarm* insert used in these experiments since in other instances where only a Wingless signal is required, such as the induction of the wing primordium during the early events of wing development, overexpressing Arm* or Wingless has very similar effects. A possible insight into the differences that the expression of Wingless and Arm* has on neurogenesis comes from the experiments where these two proteins are coexpressed with the ECN molecule. In these experiments the phenotypes generated by expressing UASECN with UASwg or UASarm* are very similar; namely, disrupting Notch signaling by expressing the ECN protein makes UASarm* and UASwg functionally equivalent. This suggests that the difference between the phenotypes generated by expressing Wingless and Arm* on their own might arise from the ability of Wingless to inhibit Notch signaling, which Arm* is unable to do; attenuating Notch signaling blocks lateral inhibition, which leads to increased numbers of sense organs. Since Wingless can activate Armadillo, overexpression of Wingless can achieve both effects simultaneously (Brennan, 1999).

When Arm* is coexpressed with ECN, the dominant negative molecule reduces Notch signaling, providing the function of Wingless that is missing in Arm* and thus making this molecule functionally equivalent to Wingless. These results raise the question of how Wingless signaling inhibits Notch signaling and where in the Wingless signaling pathway the cross-talk between the two pathways occurs. The inability of Arm* to inhibit Notch signaling indicates that the cross-talk must occur upstream of Armadillo. One possibility is that the inhibition occurs through Wingless interacting with the extracellular portion of Notch, preventing the Notch protein from interacting with its ligands. However, it is more likely to occur through the interaction of Dishevelled with the intracellular domain of the Notch protein, which has been shown previously to inhibit Notch signaling (Axelrod, 1996). In keeping with this, it has been found that overexpressing the Dishevelled protein can induce sense organ development as effectively as overexpressing Wingless; this suggests that Dishevelled can also disrupt Notch signaling as effectively as Wingless. Finally, it is possible that the interaction of Notch with both Dishevelled and Wingless is required to inhibit Delta signaling through Notch, since it has been shown previously that the ability to overexpress Dishevelled, which induces supernumerary sense organs, requires Wingless function (Axelrod, 1996). The interference of Wingless signaling with Notch signaling can also provide an explanation for the effects of ectopic expression of Wingless on the patterning of the veins and its sensitivity to the concentration of Delta. Overexpression of Wingless would reduce the availability of Notch for lateral inhibition by causing Dishevelled to sequester Notch into complexes that are unable to transduce the Delta signal. This would reduce the effectiveness of lateral inhibition signaling, an effect which would be exaggerated in situations of limiting signaling, as is observe in Dl heterozygotes or when Wingless is coexpressed with ECN (Brennan, 1999).

The interaction of Wingless and Notch signaling that has been observed might also be important during normal neural development. Wingless and Delta have opposite effects during neurogenesis; Wingless promotes while Delta suppresses the development of sense organs. Various experiments suggest that during the segregation of neural precursors a reduction of Notch signaling in the precursors themselves is as important as the Delta-mediated activation of Notch signaling in the surrounding cells. It is possible that, like the activation of Notch by Delta, the suppression of Notch signaling is an active process mediated by the interaction of Wingless and Dishevelled with Notch. If this were the case, since both Delta and Wingless have spatially and temporally regulated patterns of gene expression, their interactions with Notch could contribute to the well-documented bias in the appearance of precursors from clusters of cells with neural potential. This competitive interaction could also account for the observed increases in Wingless signaling associated with reductions in Notch signaling during lateral inhibition (Brennan, 1999).

Analysis of the mechanisms that control epithelial polarization has revealed that cues for polarization are mediated by transmembrane proteins that operate at the apical, lateral, or basal surface of epithelial cells. Whereas for any given epithelial cell type only one or two polarization systems have been identified to date, the follicular epithelium in Drosophila ovaries uses three different polarization mechanisms, each operating at one of the three main epithelial surface domains. The follicular epithelium arises through a mesenchymal-epithelial transition. Contact with the basement membrane provides an initial polarization cue that leads to the formation of a basal membrane domain. Moreover, mosaic analysis was used to show that Crumbs (Crb) is required for the formation and maintenance of the follicular epithelium. Crb localizes to the apical membrane of follicle cells that is in contact with germline cells. Contact to the germline is required for the accumulation of Crb in follicle cells. Discs lost (Dlt: now redefined as Drosophila Patj), a cytoplasmic PDZ domain protein that has been shown to interact with the cytoplasmic tail of Crb, overlaps precisely in its distribution with Crb, as shown by immunoelectron microscopy. Crb localization depends on Dlt, whereas Dlt uses Crb-dependent and -independent mechanisms for apical targeting. The cadherin-catenin complex is not required for the formation of the follicular epithelium, but only for its maintenance. Loss of cadherin-based adherens junctions caused by armadillo mutations results in a disruption of the lateral spectrin and actin cytoskeleton. Also Crb and the apical spectrin cytoskeleton are lost from armadillo mutant follicle cells. Together with previous data showing that Crb is required for the formation of a zonula adherens, these findings indicate a mutual dependency of apical and lateral polarization mechanisms (Tanentzapf, 2000).

The cadherin-catenin complex plays a major role in epithelial polarization because cadherin-mediated adhesive contacts cause the assembly of the lateral surface domain. Consequently, lack of cadherin activity compromises epithelial integrity in many tissues or epithelial cell culture lines. Removal of E-cadherin (Shotgun) from follicle cells causes only mild defects in the development of the FE. In follicle cells that lack E-cadherin, some Arm is retained at adherens junctions, raising the possibility that the FE coexpresses a second cadherin that interacts with Arm. Indeed, N-cadherin is expressed in follicle cells in a pattern that overlaps with E-cadherin in early- to mid-oogenesis. N-cadherin disappears from the FE at stage 10 of oogenesis, whereas E-cadherin is expressed throughout oogenesis. In contrast to E-cadherin, N-cadherin is not expressed in the cells of the germline. However, Arm appears to be the only ß-catenin homolog in Drosophila, in contrast to vertebrates, where ß-catenin can be functionally replaced by plakoglobin in the cadherin-catenin complex. Thus, to effectively remove the cadherin-catenin complex from follicle cells, clones were generated that lack Arm, which is known to interact with both E- and N-cadherin (Tanentzapf, 2000).

Clones were induced for three different mutant arm alleles that carry premature stop codons in the 6th (armYD35), 7th (armXK22), and 10th (armXP33) arm repeat. armXP33 is an intermediate hypomorph; armXK22 is a strong hypomorph, and armYD35 is believed to be a null allele. Embryos derived from germline clones mutant for armXP33 show a dramatic disruption of epithelial morphology that occurs at the onset of gastrulation and is substantially more severe than the defects in epithelial structure seen in crb null mutant embryos. Moreover Crb is needed for the formation of the ZA in embryonic epithelia. If the failure to form the ZA is the major consequence of compromising Crb or Dlt activity in the FE, the lack of the cadherin-catenin complex would be expected to cause similar defects as seen in crb and dlt mutant follicle cells; that is, a failure to form a FE. Surprisingly, follicle cells mutant for any of the three arm alleles form a FE. No follicles were observed in these experiments that show epithelial discontinuities, as seen in crb and dlt mutant follicles. arm mutant follicle cells often show an irregular morphology at early stages of follicle development. The irregularities in epithelial structure increase in severity until the FE is compromised and the follicle degenerates at mid- to late-oogenesis. To determine whether adherens junctions were effectively disrupted in arm mutant follicle cells, the expression of E- and N-cadherin was examined in those cells. Neither E- nor N-cadherin are detected in follicle cells mutant for any of the three arm alleles studied. Taken together, these findings suggest that cadherin-based adherens junctions are not essential for the formation of the FE, but are important for maintaining its epithelial structure (Tanentzapf, 2000).

Advantage was taken of the fact that arm mutant cells in the FE are maintained for several days and their molecular architecture was examined. armXP33 mutant follicle cells, which in most cases have a normal cuboidal to columnar shape, show a decrease of F-actin and alpha-spectrin at the lateral membrane, and an accumulation of these molecules at the apical cell pole. In contrast, the apical marker ßHeavy-spectrin shows a normal distribution in armXP33 mutant cells, suggesting that the apical spectrin cytoskeleton is intact. Follicle cells mutant for armXK22 or armYD35 often develop a squamous cell morphology or show a multilayered structure. alpha-Spectrin remains associated with the narrow lateral membranes in squamous arm mutant cells. ßHeavy-Spectrin, in contrast, is lost from these follicle cells, suggesting that the apical spectrin cytoskeleton is disrupted. To further examine the apical surface domain of arm mutant follicle cells, the distribution of Crb and Dlt was examined in these cells. armXP33 mutant follicle cells typically show a normal apical localization of Crb and Dlt. In contrast, Crb is lost from the apical membrane of follicle cells mutant for strong arm alleles, whereas apical Dlt is retained in these cells. Similar to dltdre1 mutant cell clones, Dlt forms a cap in the center of the apical membrane of arm mutant follicle cells. These observations suggest that the disruption of adherens junctions leads to a breakdown of the lateral membrane domain, as expected, but that this also compromises the apical surface domain. The differential behavior of Crb and Dlt in strong arm mutant cell clones again emphasizes that Dlt can rely on a Crb-independent apical targeting mechanism, and shows that apical Dlt can be retained in the absence of an apical spectrin cytoskeleton (Tanentzapf, 2000).

A screen for mutations that suppress the phenotype of armadillo

During development, signaling pathways coordinate cell fates and regulate the choice between cell survival or programmed cell death. The well-conserved Wingless/Wnt pathway is required for many developmental decisions in all animals. One transducer of the Wingless/Wnt signal is Armadillo/ß-catenin. Drosophila Armadillo not only transduces Wingless signal, but also acts in cell-cell adhesion via its role in the epithelial adherens junction. While many components of both the Wingless/Wnt signaling pathway and adherens junctions are known, both processes are complex, suggesting that unknown components influence signaling and junctions. A genetic modifier screen was carried out to identify some of these components by screening for mutations that can suppress the armadillo mutant phenotype. Twelve regions of the genome were identified that have this property. From these regions and from additional candidate genes tested, four genes were identified that suppress arm: dTCF, puckered, head involution defective (hid), and presenilin. The interaction with hid, a known regulator of programmed cell death, was further investigated. The data suggest that Wg signaling modulates Hid activity and that Hid regulates programmed cell death in a dose-sensitive fashion (Cox, 2000).

It has been known for more than a decade that PCD plays an important role in the segment polarity phenotype resulting from inactivation of either the Hedgehog or Wg pathways. Detailed analysis of this process has been carried out, quantitating cell death in wg, arm, gooseberry, and naked. The elevation in cell death affects particular cells. Since the first reports of cell death in segment polarity mutants, the machinery that drives PCD in embryos has begun to be identified. Homozygosity for the small chromosomal Deficiency, Df(3L)H99, blocks essentially all PCD. Within this interval, three genes play roles in PCD: grim, reaper, and hid. Ectopic expression of any of these can trigger PCD, but loss-of-function mutations are only available for hid (Cox, 2000 and references therein).

Given the role of PCD in the segment polarity phenotype, it is perhaps not surprising that elimination of PCD would alter it. Several aspects of the effect of PCD reduction were unexpected, however. First, and most striking, the phenotypes of arm and wg mutants were very sensitive to relatively small changes in the dose of hid and the other cell-death promoters. For example, while heterozygosity for hid has no known effects on normal development, it strongly suppresses arm. Further reductions in the levels of hid or the other cell-death regulators have no additional effect on arm, suggesting that reducing the Hid dose by half eliminates the relevant ectopic PCD that occurs in an arm mutant. The wg phenotype is also suppressed in a highly dose-sensitive fashion, but in a different dosage range. A 50% reduction of hid causes slight but detectable effects; a 50% reduction in all three death promoters causes greater suppression, while homozygosity for the deletion removing all three genes results in the strongest wg suppression (Cox, 2000).

Recent observations regarding the role of Hid in PCD in the eye may explain this. Signaling through the ras/mitogen-activated protein kinase (MAPK) pathway promotes cell survival by antagonizing Hid. It has been suggested that Hid serves as a rheostat, with its levels determining the probability of PCD. It has been further suggested that Hid activity has to exceed a threshold to trigger PCD; the accumulation of hid mRNA in cells that are not programmed to die is consistent with this. Current observations further support this model. Wg signaling may normally antagonize Hid, potentially by regulating its expression. In embryos where Wg signaling is attenuated, elevated Hid activity may trigger PCD when it rises above a critical threshold. A threshold model could explain why the segment polarity phenotype is so sensitive to the dose of Hid and its partners (Cox, 2000 and references therein).

Another surprise was the qualitative difference in the effect of cell death reduction on wg and arm mutants. While the resulting cell number is likely increased in both double-mutant genotypes in the arm; hid double mutant, the reduction in PCD restored an almost wild-type-length cuticle, while in the wg;hid double mutant, the increase in cell number is not reflected in an increase in cuticle length. The reason for this remains a mystery. One possible explanation for this discrepancy is the difference in the degree to which Wg signal is compromised in the two situations and the embryonic stage at which this disruption occurs. In the wg null, Wg signaling is totally eliminated from the beginning of development. In contrast, perdurance of maternal Arm substantially rescues early defects in Wg signaling in arm zygotic nulls. arm mutants remain more normal in morphology than wg mutants through the onset of germband retraction and retain remnant denticle diversity. Thus when one eliminates PCD in an arm mutant a more normal pattern is restored. The difference in amount and timing of Wg signaling in the two backgrounds may also explain why arm mutants are affected by smaller alterations in Hid level. The remaining Wg signaling in an arm zygotic mutant may promote cell survival to some extent, meaning that a smaller reduction in Hid activity prevents ectopic PCD (Cox, 2000).

It is also surprising that reduction in cell death alleviates arm's dorsal closure defect. It has been suspected that this defect is due solely to Arm's role as a catenin. However, recent data suggest that dorsal closure is promoted by Wg signaling. It is now suspected that defects in Wg signaling and catenin function combine to block dorsal closure in arm mutants. Restoring either rescues the arm dorsal closure defect. However, blocking PCD alone should not restore Wg signaling or catenin function. Perhaps the excess cell death in the head region or in the amnioserosa of an arm mutant contributes to its dorsal closure defect (Cox, 2000).

While evaluating the effectiveness of this screen, a variety of candidate genes, including some that map within noninteracting Deficiencies, were tested. Heterozygosity for one of these, Presenilin, strongly suppresses arm. Both ßcat and other Arm repeat proteins such as delta-catenin associate with Presenilins in vivo. The function of this interaction remains confusing. Wild-type Presenilin stabilizes ßcat; this stabilization is abrogated by missense mutations in presenilin, and presenilin missense mutant cells from mutant patients have less nuclear ßcat. These data support a role for Presenilins as positive regulators of Wnt signaling via Arm/ßcat. In contrast, it has been reported that overexpression of wild-type Presenilin destabilizes ßcat; ßcat is stabilized in both Presenilin1 null fibroblasts or if Presenilin1 mutations are overexpressed, while a Wnt-responsive promoter is downregulated by Presenilin overexpression. These data support a conclusion that is the opposite of the one above, in which wild-type Presenilins negatively regulate Wnt signaling. It has been suggested that the presenilin-ßcat complex includes cadherins, in contravention of most other data. The genetic data are most consistent with a model in which Presenilins negatively regulate Wg signaling either directly or indirectly by binding Arm/ßcat or by influencing adherens junction assembly. Clearly much work remains to differentiate between the different possible mechanisms (Cox, 2000 and references therein).

Armadillo, Nemo, the Wingless pathway and apoptosis

Nemo-like kinases define a novel family of serine/threonine kinases that are involved in integrating multiple signaling pathways. They are conserved regulators of Wnt/Wingless pathways, which may coordinate Wnt with TGF-mediated signaling. Drosophila nemo was identified through its involvement in epithelial planar polarity, a process regulated by a non-canonical Wnt pathway (see Habas and Dawid Dishevelled and Wnt signaling: is the nucleus the final frontier?). Ectopic expression of Nemo using the Gal4-UAS system results in embryonic lethality associated with defects in patterning and head development. An analyses of nemo phenotypes of germline clone-derived embryos is described. Lethality is observed associated with head defects and reduction of programmed cell death and it is concluded that nemo is an essential gene. Data is presented showing that nmo is involved in regulating apoptosis during eye development, based on both loss of function phenotypes and on genetic interactions with the pro-apoptotic gene reaper. Genetic data from the adult wing are presented that suggest the activity of ectopically expressed Nemo can be modulated by Jun N-terminal kinase (JNK) signaling. Such an observation supports the model that there is cross-talk between Wnt, TGFß and JNK signaling at multiple stages of development (Mirkovic, 2002).

Nemo proteins have been found to play a role in regulating Wnt signaling. Thus, it is interesting that characterization of segment polarity mutants revealed that both wg and arm mutant embryos have elevated levels of cell death. These findings imply that those gene products normally act to inhibit cell death at least during embryogenesis. Further evidence that Wnt signaling in Drosophila acts to promote cell viability comes from a genetic screen in which dominant modifiers of arm were identified. In this screen, genes were identified that could rescue the severe armXP33 embryonic cuticle phenotype. Heterozygosity for hid shows a dominant suppression of aspects of the phenotype, suggesting that at least some of the arm phenotypes are due to excess apoptosis. Furthermore, expression of the baculoviral caspase inhibitor p35 in the arm mutant background also rescued the phenotype, establishing that the rescue observed in both cases was due to lowering the amount of apoptosis. These data which imply that Wnt signaling opposes apoptosis, combined with the finding that Nemo promotes apoptosis, suggest a possible mechanism whereby Nemo can promote cell death by inhibiting Wnt signaling (Mirkovic, 2002).

EGF receptor/Rolled MAP kinase signalling protects cells against activated Armadillo in the Drosophila eye

ß-catenin/Armadillo are transcriptional co-activators that mediate Wnt signalling in normal development. Activated forms of ß-catenin are oncogenic. Mutant forms of Drosophila Armadillo were constructed that correspond to common human oncogenic mutations; they were found to activate Armadillo constitutively. When expressed in the Drosophila eye, these eventually induce apoptosis in all cell types. Intriguingly, cells in the eye are resistant to the effects of activated Armadillo for a long period prior to the onset of cell death at the mid-pupal stage. This latency is conferred by EGF receptor (EGFR)/MAP kinase signalling, which prevents activated Armadillo from inducing apoptosis; when EGFR signalling naturally ceases, the cells rapidly die. Nemo, the Drosophila homologue of NLK in mice and LIT-1 in Caenorhabditis elegans, does not antagonize activated Armadillo, suggesting that the Nemo-like MAP kinases may not generally interact with Armadillo/ß-catenin. Thus, the results show that activated Armadillo is subject to a specific negative control by EGFR/Rolled MAP kinase signalling (Freeman, 2001).

Expression of activated forms of Armadillo causes a late onset of apoptosis in the developing Drosophila eye. These effects are similar to those of overexpressing dTCF, and genetic interactions indicate that they are mediated by endogenous dTCF. The disruptive effects of these conditions closely mimic those of Drosophila APC mutations except that, since APC expression is restricted to neuronal cells, cell death in that case is confined to photoreceptors. Therefore, in flies as in humans, the consequences of activated Armadillo/ß-catenin are essentially equivalent to those caused by APC loss. Importantly, these new forms of activated Armadillo provide powerful tools for genetic screens which might identify ancillary signalling proteins conserved between humans and Drosophila (Freeman, 2001).

A precedent for an antagonistic effect between Wingless and EGFR signalling is found in the embryonic epidermis. In this case, signalling by these pathways appears to be integrated at the level of a common target gene, svb. Given that, in the eye disc, overexpression of dTCF causes the same delayed cell death as Arm*, and that the Arm* rough eye phenotype is suppressed by dTCF heterozygosity, the antagonism between Arm* and EGFR signalling also occurs at the level of target gene transcription. As in the embryonic epidermis, antagonistic inputs from the two pathways could be integrated at a transcriptional enhancer of a common target gene. However, svb is not a good candidate in this case since it is not expressed in the eye and no genetic interaction between svb and Arm* has been found (Freeman, 2001).

Although it has been argued that the delayed apoptotic effect of Arm* is based primarily on dTCF-mediated transcription, there may also be a post-transcriptional contribution. This is suggested by the observation that Armadillo levels were only moderately elevated in larval eye discs overexpressing Arm*, but strongly increased in 40-48-h-old pupal discs that showed severely disrupted ommatidia. As was observed for Armadillo S10, both cytoplasmic and nuclear levels were increased simultaneously, indicating that both types of activating mutations cause stabilization of free protein rather than altering its relative distribution between cytoplasm and nucleus. However, since this increase in Armadillo level correlates in time with the onset of apoptosis, it is unclear whether the accumulation is a cause or an effect of the putative transcriptional changes induced by Arm* (Freeman, 2001).

Most interestingly, in contrast to other tissues where activated Armadillo has immediate effects, Arm* is expressed for 2-4 days in eye disc cells before causing a detectable phenotype; onset of apoptosis occurs only at the mid-pupal stage. This delayed manifestation of the effects of Arm* could not have been inferred from the phenotype of dAPC mutants. These results imply that this latency is a consequence of protection conferred by EGFR signalling. The EGFR has multiple functions in the developing eye, including a protective effect against cell death in normal development. Furthermore, a strong genetic interaction has been observed between the cell death-inducing factor Hid and Ras1 signalling in the eye, and the latter apparently promotes cell survival by downregulating Hid directly at the transcriptional and post-transcriptional level (Freeman, 2001).

In contrast, no suppression is seen of the Arm* rough eye phenotype by heterozygosity for the H99 deficiency, which uncovers Hid and two further cell-death-inducing factors. This argues that the antagonism observed between Arm* and EGFR signalling is specific, and distinct from the more general survival-promoting function of the receptor, which is mediated directly by Hid. In support of this, a large number of genes including dE2F, p53 and presenilins have been found to induce cell death when expressed in the larval eye disc; in all cases, the apoptosis occurs in the third instar disc, despite the fact that normal EGFR signalling is very active at this stage. In the case of dE2F, this early-onset apoptosis is antagonized by EGFR signalling, reflecting the ongoing survival-promoting role of the EGFR throughout disc development. The distinction between this and the phenomenon report in this study is demonstrated by the fact that the rough eye phenotype caused by simultaneous overexpression of dE2F, dDP and p35 is not suppressed by dTCF or arm heterozygosity. Also, there is no known requirement for arm in the larval disc. Taken together, these observations suggest that the dramatic EGFR-mediated delay of the Arm*-induced apoptotic phenotype is unique, and that the mechanism on which it is based is distinct from that employed by the receptor in generally promoting cell survival in the disc (Freeman, 2001).

Armadillo/ß-catenin-dependent Wnt signalling is required for the polarisation of epidermal cells during dorsal closure in Drosophila

At the end of germband retraction, the dorsal epidermis of the Drosophila embryo exhibits a discontinuity that is covered by the amnioserosa. The process of dorsal closure (DC) involves a coordinated set of cell-shape changes within the epidermis and the amnioserosa that result in epidermal continuity. Polarization of the dorsal-most epidermal (DME) cells in the plane of the epithelium is an important aspect of DC. The DME cells of embryos mutant for wingless or dishevelled exhibit polarization defects and fail to close properly. The role of the Wingless signalling pathway in the polarization of the DME cells and DC was investigated. The ß-catenin-dependent Wingless signalling pathway is required for polarization of the DME cells. Although the DME cells are polarized in the plane of the epithelium and present polarized localization of proteins associated with the process of planar cell polarity (PCP) in the wing, e.g., Flamingo, PCP Wingless signalling is not involved in DC (Morel, 2004).

The initiation of DC in Drosophila embryos correlates with the elongation and polarization of the DME cells in the DV axis of the embryo. In parallel with this polarization, a cable of F-actin assembles on the dorsal-most surface of these cells and promotes the formation of filopodia and lamellae during the final phases of the process. A functional link between the polarization and the assembly of the cable of actin is supported by the observations that in mutants in which the DME cells do not elongate, there is no actin cable and no dynamic protrusions. As a consequence, these embryos display defects and delays in the closure process. Embryos mutant for wg and dsh are good examples of this class (Morel, 2004).

The polarization of the DME cells occurs in the plane of the epithelium and can be seen as a manifestation of the phenomenon of planar cell polarity (PCP). Since a specific branch of Wg signalling has been implicated in PCP and there is evidence for an interaction between Dishevelled and JNK signalling during dorsal closure, whether there is a role for this mode of Wg signalling in the process of DC was tested. The results clearly show that the 'canonical' Wg signalling pathway that leads to activation of Armadillo and of the transcription of target genes is necessary and sufficient to restore the polarity of the DME cells and to promote a normal process of dorsal closure in a wg mutant embryo. Surprisingly, it was found that the PCP pathway does not appear to play a major role in DC or the polarization of the DME cells, since activation of the 'canonical' pathway in the absence of dsh activity rescues the polarity and function of the DME cells.

Dsh contains three highly conserved domains, the DIX, PDZ and DEP domains. The DEP domain mediates interaction of Dsh with the cell cortex and is required for PCP but not 'canonical' Wg signalling, while the DIX domain is required for the 'canonical' Wg signalling but seems dispensable for PCP. To investigate an involvement of the PCP pathway in the activities of the DME cells during DC, rescue experiments of wg embryos were carried out using truncated forms of Dsh deleted for either the DEP (DshDeltaDEP) or the DIX (DshDeltaDIX) domain. Although overexpression of DshDeltaDEP leads to the partial rescue of naked cuticle and of En expression, neither naked cuticle nor rescue of En expression are observed in wg>da>DshDeltaDIX (using da-GAL4 to drive DshDeltaDIX in wg mutants) embryos. This thus confirms that DshDeltaDEP is able to signal within the 'canonical' Wg pathway but not DshDeltaDIX (Morel, 2004).

Then the ability of either protein to rescue DC in wg embryos was tested. wg>da>DshDeltaDEP embryos are longer than wg mutants and their dorsal cuticle is improved; no hole is observed and only occasional warts can be seen. The DME cells are oriented in the DV direction and most of them show a slight elongation in the DV direction when the zippering process has started. Simultaneously, Fmi is observed at the membrane and accumulates at the level of the ANCs. Although no clear elongation of DME or ventral epidermal cells is observed, DC process is improved; two zippers, at the anterior and posterior ends of the embryo, are initiated, whereas only the posterior one is observed in wg embryos. By contrast, wg>da>DshDeltaDIX embryos have a shorter cuticle than wg mutants and show a more severe puckering and hole on the dorsal side. Furthermore, neither the shape nor the polarization of DME cells is improved in these embryos (Morel, 2004).

Thus, although DshDeltaDEP can rescue partially the DC defects of wg mutants, ubiquitous overexpression of DshDeltaDIX does not rescue any of the observed features confirming the requirement for the Wg 'canonical' pathway during DC. Thus, the conclusion that the PCP pathway does not appear to play a role in DC or in the polarization of the DME is supported by the observation that although a moiety of Dishevelled that promotes Armadillo signalling is capable of rescuing the defects of wg mutants, a moiety that promotes JNK signalling and PCP does not. Altogether, these results indicate that the polarization and activity of the DME cells during dorsal closure requires Armadillo/ß-catenin-dependent Wg signalling. Furthermore, this requirement is restricted to the epidermis because activation of Wg signalling in the amnioserosa has no effect on the epidermis (Morel, 2004).

The polarization of the DME cells and subsequent dynamics of actin at the LE can be construed since the development of the leading edge of a motile cell and to a certain extent is akin to an epidermal/mesenchymal transition (EMT), as one of the features of this process is the reorganization of the actin cytoskeleton and the acquisition of motility by the cells. In this regard, it is interesting to note that ß-catenin-dependent Wnt signalling has been implicated in EMT both in normal and cancerous cells and that therefore there are precedents for the involvement of the ß-catenin-mediated transcriptional regulation in the development of actin dynamics. However, the targets of the Wnt pathway mediating this process are not known (Morel, 2004).

It has been suggested that Dpp is a central effector of dorsal closure. Embryos mutant for dpp signalling exhibit defects in dorsal closure. dpp is expressed in the DME cells and has been proposed to act as a long range signal for the elongation of the more ventral cells. Wingless is shown to be required for the correct maintenance of dpp expression in the DME cells, although in these experiments the input is less significant than has been reported before. Altogether, these observations suggest that some of the activity of Wingless during DC is mediated by Dpp. Indeed, when the Dpp pathway was ubiquitously activated by the means of an activated form of its receptor Tkv, some rescue of the polarity of the DME cells was observed. However, although in this case the DME cells orient themselves in the DV direction and Fmi localises as it does in wild type, neither the DME nor the ventral epidermal cells elongate, and the DC process is not substantially improved. This contrasts with the full rescue of both the polarization of DME cells and the DC process following ubiquitous activation of the ß-catenin-dependent Wg pathway. Thus, if Dpp contributes to DC, it is not as the only target of Wg signalling (Morel, 2004).

Expression of Wingless from the amnioserosa in wg mutants induces high and continuous levels of dpp in the DME cells together with some rescue of the polarity of the DME cells but without any effect on the elongation of these or the more ventral cells. This rescue is very similar to the one observed with ubiquitous expression of the activated Tkv. These results indicate that Dpp does not act as a long-range signal for the elongation of the more ventral epidermal cells; rescue of Dpp expression in the DME cells or activation of Dpp signalling throughout the epidermis in wg mutants does not lead to the elongation of the more ventral cells. A similar conclusion had been suggested from the observation that epidermal cells initially elongate in the absence of Dpp signalling but resume their polygonal shape soon after. However, an alternative explanation for these observations is that the elongation of the ventral epidermal cells requires inputs from both Dpp and Wingless signalling (Morel, 2004).

Altogether, these observations indicate that Dpp is not the only effector of Wingless during DC and indicates that Wingless signalling via Armadillo controls genes that act either in parallel or together with those regulated by JNK and Dpp (Morel, 2004).

Wnt signaling is required for long-term memory formation

Wnt signaling regulates synaptic plasticity and neurogenesis in the adult nervous system, suggesting a potential role in behavioral processes. This study probed the requirement for Wnt signaling during olfactory memory formation in Drosophila using an inducible RNAi approach. Interfering with β-catenin expression in adult mushroom body neurons specifically impairs long-term memory (LTM) without altering short-term memory. The impairment is reversible, being rescued by expression of a wild-type β-catenin transgene, and correlates with disruption of a cellular LTM trace. Inhibition of wingless, a Wnt ligand, and arrow, a Wnt coreceptor, also impairs LTM. Wingless expression in wild-type flies is transiently elevated in the brain after LTM conditioning. Thus, inhibiting three key components of the Wnt signaling pathway in adult mushroom bodies impairs LTM, indicating that this pathway mechanistically underlies this specific form of memory (Tan, 2013).

This study was prompted by a previous discovery that a casein kinase Iγ homolog (CkIγ), gilgamesh (gish), is required for STM in Drosophila (Tan, 2010). CkIgγmediated phosphorylation of the cytoplasmic tail of Lrp5/6 (Arr) is crucial for Wnt/β-catenin signaling (Davidson, 2005), and it was predicted that disruption of the Wnt signaling pathway would perturb STM. Surprisingly, however, it was found that knockdown of the four Wnt signaling components leaves STM intact. The likely explanation for this discrepancy is that Gish serves other important functions in STM formation besides its role in LTM through phosphorylation of the Arr receptor (Tan, 2013).

How does Wnt signaling in the MB neurons mediate the formation of LTM? Since the normal expression of β-catenin, Wg, and Arr is required in the set of MB neurons defined by P{MB-GeneSwitch}12-1, and Wg is a short-range ligand, a model is favored in which the Wnt ligand, Wg, participates in an autocrine fashion in the MB neurons. Spaced conditioning, which produces long-term behavioral memory, but not massed or single-cycle conditioning, leads to a transient increase in wg expression in the MB neurons, perhaps as a step downstream of Creb. The subsequent secretion of Wg by the MB neurons activates the Fz/Arr receptor, leading to the accumulation of β-catenin in the MB neurons. β-catenin, in turn, orchestrates transcriptional changes in the MB neurons that are required for LTM, as well as the breaking and remaking of cell contacts through N-cadherin function, which is necessary for the reorganization of synapses for LTM storage. Recently, ribonucleoprotein particles containing synaptic protein transcripts were shown to exit the nucleus through a nuclear envelope budding process in response to Wnt signaling at the Drosophila neuromuscular junction (Speese, 2012). Wnt-dependent nuclear budding could provide the initial step for transporting RNAs to synapses for local protein synthesis and LTM formation (Tan, 2013).

back to armadillo Effects of Mutation part 1/2

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

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.