puckered

DEVELOPMENTAL BIOLOGY

Embryonic

Puckered RNA is apparent in early embryos (0-4 hr after egg laying [AEL]) suggesting the presence of maternal transcripts. Puc mRNA expression was detected only in a small number of the experiments, which suggests a very low level of expression or transcript instability. Expression of PUC mRNA is observed in the dorsal-most cells at the leading edge of the epidermis. This pattern is identical to that described for the early expression of beta-galactosidase in the insertional alleles (Ring, 1993). After stage 11, PUC mRNA expression slowly decays on the leading edge, whereas beta-galactosidase is found up to completion of dorsal closure (Ring, 1993).

Pupal stage

Dorsal closure, a morphogenetic movement during Drosophila embryogenesis, is controlled by the Drosophila JNK pathway, D-Fos and the phosphatase Puckered (Puc). To identify principles of epithelial closure processes, another cell sheet movement that can be termed thorax closure was studied: the joining of the parts of the wing imaginal discs that gives rise to the adult thorax during metamorphosis. The genes that are required for dorsal closure give rise to an interesting abnormal adult phenotype, suggesting that there is an additional requirement for these genes during later development: homozygous animals of mutant alleles of D-fos, hep, pannier (pnr) and components of the Dpp pathway show a cleft at the dorsal midline of the thorax and neighbouring bristles are abnormally parted to both sides. In thorax closure a special row of margin cells express puc and accumulate prominent actin fibers during midline attachment. Genetic data indicate a requirement for D-Fos and the JNK pathway for thorax closure, and a negative regulatory role for Puc. Furthermore, puc expression co-localizes with elevated levels of D-Fos. It is reduced in a JNK or D-Fos loss-of-function background, and is ectopically induced after JNK activation. This suggests that Puc acts downstream of the JNK pathway and D-Fos to mediate a negative feed-back loop. Therefore, the molecular circuitry required for thorax closure is very similar to the one directing dorsal closure in the embryo, even though the tissues are not related. This finding supports the hypothesis that the mechanism controlling dorsal closure has been co-opted for thorax closure in the evolution of insect metamorphosis and may represent a more widely used functional module for tissue closure in other species as well (Zeitlinger, 1999).

In order to mark and visualize the dorsal parts of the wing imaginal discs that fuse during thorax closure, the UAS-Gal4 system was used to express green fluorescent protein in the expression domain of pnr, a gene encoding a GATA transcription factor whose expression is restricted to dorsal tissues throughout development. The prepupae were then dissected in a way that leaves the entire thorax complex intact and different stages were inspected by confocal microscopy. In addition, actin filaments were visualized by staining with phalloidin to monitor the behaviour of the cytoskeleton during this process. Phalloidin also stains three oblique muscles on each side, a useful marker during thorax closure. Already in third instar wing imaginal discs, pnr expression marks the dorsal part, the future medial notum. At around 6 hours after pupation (AP), after eversion, the dorsal parts of the two wing imaginal discs spread toward the dorsal midline, while the larval epidermis degenerates. When they subsequently meet and attach to each other at around 7 hours AP, filamentous actin becomes visible at the medial edge of the epithelium. These actin bundles at the dorsal midline are most abundant at 8 hours AP and are predominantly localized basally (Zeitlinger, 1999).

In summary, the process of thorax closure resembles embryonic dorsal closure at a tissue-morphological level: two epithelial sheets with a straight margin approach one another, meet at the dorsal midline, and attach. The actin organization seen along the margin of the epithelium is reminiscent of the accumulation of actin along the leading edge of the closing embryo. However, in contrast to the simple epithelial stretching of embryonic dorsal closure, the morphogenetic movements involved in thorax closure appear to be more complex: most cells are of polygonal shape and not obviously elongated along the dorsoventral axis. Furthermore, the tissue movements also include unfolding (as part of the eversion) and an anterior folding-in during midline fusion with subsequent back folding during head eversion (Zeitlinger, 1999).

Having established a system to monitor the progress of thorax closure, the tissue movements were monitored in a mutant background that gives rise to a cleft phenotype in adults. The hypomorphic mutation in D-fos, kay ² was used in this experiment. It revealed that the dorsomedialward spreading of the epithelium is already abnormal at 6 hours AP in most kay² prepupae. While, in a wild-type background, the pnr expression domain of the wing imaginal disc is found on top of the three oblique muscles and close to the degenerating larval epidermis, the corresponding epithelium in kay² prepupae of this stage has failed to reach this position and is still located more laterally. At 8 hours AP, the spreading epithelium often appears to have retracted and fallen back into its original folded position found at earlier stages, although filamentous actin typical of this stage is detectable. These findings strongly argue that the defects observed in kay² adult animals result from defects in thorax closure during prepupal stages (Zeitlinger, 1999).

The thoracic cleft phenotype observed with hypomorphic mutations in D-fos (kay² ) and hep (hep¹) suggests that D-Fos and the JNK pathway are involved in thorax morphogenesis. To confirm that the cleft phenotype is a result of a D-fos loss-of-function condition, a dominant negative form of D-fos (UAS-D-Fos bZIP) was expressed under the control of pnr-Gal4. This results in the appearance of a marked cleft in the thorax. A similar phenotype is obtained by overexpressing Puc (UAS-Puc) in the pnr domain. In the embryo, overexpression of Puc phenocopies loss-of-function mutations in the JNK pathway, consistent with the proposed function of Puc as a phosphatase that negatively regulates the JNK pathway by dephosphorylation of Basket. The fact that this is also true in thorax closure represents further evidence that the JNK pathway is involved in thorax closure. Next, a test was performed to see whether D-Fos genetically interacts with components of the JNK pathway during thorax closure. In contrast to the D-fos hypomorphic mutant kay², kay¹ represents a D-fos null allele (a deficiency). The heterozygous allelic combination (kay¹ / kay²) is strictly lethal, but can be rescued by ubiquitous expression of D-Fos under a heterologous promoter. Strikingly, the lethality of kay¹ / kay² could also be rescued by eliminating one copy of the wild type puc gene. More than 50% of the expected Mendelian frequency could be recovered. Thus, puc^E69 has a dominant effect in a kay mutant background, even though heterozygosity for puc^E69 has no phenotypic effects in an otherwise wild-type fly. Furthermore, not only the lethality but also the thorax cleft phenotype of kay mutant flies could be dominantly rescued. The cleft phenotype of the rescued kay² / kay¹ puc flies ranges from strong to very mild. Heterozygous puc^E69 in a kay² homozygous background (kay² / kay² puc E69 ) gives rise to a stable stock in which most flies show a very mild or no thorax cleft at all. Therefore, the puc mutation has a dominant effect on thorax closure and two conclusions can be drawn: (1) Puc must be expressed during thorax closure; (2) as in dorsal closure, Puc negatively regulates the pathway in which D-Fos is acting during thorax closure (Zeitlinger, 1999).

Effects of mutation or deletion

The shape of the dorsal-most cells is also abnormal in puc mutants. During germ band shortening these cells do not elongate as in wild type, but retain the polygonal shape observed in the extended germ band phase. Consequently, they do not generate a straight edge to the moving front of the epidermis during dorsal closure. When the two sides of the epidermis meet at the dorsal midline they do not form parallel rows. The pattern of the cuticle secreted by these cells is also abnormal (Ring, 1993).

Embryos mutant for puc develop defects along the dorsal midline of the larval cuticle during dorsal closure [see the images section]. These defects manifest as misaligned segments in the weakest allele, pucEh, and become more pronounced in stronger alleles. For example, in embryos carrying the pucE69 allele, dorsal hairs are absent along the midline, leaving a strip of naked cuticle along most of the midline; these embryos display strong puckering of the epidermis (Ring, 1993). Mutations lead to cytoskeletal defects related to defects associated with mutations in basket, the Drosophila JNK homolog. puckered mutations result in the hyperactivation of DJNK, and overexpression of puc mimics basket mutant phenotypes (Martin-Blanco, 1998).

To study if puc restrains JNK activity during the different steps of dorsal closure, the embryonic phenotype was analyzed in which puc was ectopically expressed. Embryos heat-shocked very early [between 4 and 5 hr AEL] fail to achieve dorsal closure and exhibit a large dorsal opening. Most of the embryos heat shocked between 5 and 7 hr AEL display dorsal holes or phenotypes similar to puc loss of function alleles. Heat-shocking embryos beyond 7 hr AEL produces a puc loss-of-function phenotype, and occasionally a dorsal hole that, on average, is smaller than that of embryos heat-shocked earlier. In these experiments, early overexpression of Puc mimics the phenotype of bsk mutants and the complete inactivation of DJNK signaling. Late expression of Puc affects the ability of the dorsal-most cells to differentiate properly and induces the same defects as puc loss-of-function alleles. puc in the heterozygous condition can rescue the dorsal open characteristic of low JNK signaling: hemizygous hemipterous1 embryos develop a dorsal open phenotype, which is partially rescued by pucE69 or pucA251.1 in the heterozygous condition. These results support a role of puc in limiting DJNK activity during dorsal closure and suggest the existence of a feedback loop through which JNK dependent expression of puc regulates JNK activity (Martin-Blanco, 1998).

Dorsal closure depends on the activities of a Jun amino (N)-terminal kinase kinase (JNKK) encoded by the hemipterous gene, and of a JNK encoded by basket. Hep is required for cell determination in the leading edge of migrating epithelia, by controlling specific expression of the puckered gene in these cells. puc encodes a protein related to vertebrate dual-specificity MAPK phosphatases of the CL100 family (E. Martin-Blanco, A. Gampel, and A. Martinez Arias, pers. comm. to Glise, 1997). During dorsal closure, decapentaplegic is expressed in the row of cells making up the leading edge of the epithelia. The small GTPases Dcdc42, Drac1, and the Hep JNKK control dpp expression in this migratory process. Activated Drac1 and Dcdc42 induce distinct, although partly overlapping, responses. Dcdc42 appears to be a good inducer of ectopic puc and dpp expression in ectodermal cells located more ventrally, whereas Drac1 seems more active in cells nearest to the leading edge. Appropriate dpp and puc expression in the leading edge also depends on the inhibitory function of the puc gene. puc acts as a repressor of dpp expression in the ectoderm, likely acting to inhibit Basket, the Jun N-terminal kinase. In puc mutants, ectopic expression of puc and dpp is induced in the ectoderm, that is, outside the normal domain of puc expression in the leading edge. In addition, puc (but not dpp) is expressed ectopically in amnioserosa cells. These observations indicate a cell non-autonomous effect of puc mutations. The data suggest that the leading edge is the source of a JNK autocrine signal, and exclude a role for Dpp as such a ligand. Dorsal closure couples JNK and Dpp signaling pathways, a situation that may be conserved in vertebrate development (Glise, 1997).

One of the fundamental events in insect metamorphosis is the replacement of larval tissues by imaginal tissues. Shortly after pupariation the imaginal discs evaginate to assume their positions at the surface of the prepupal animal. This is a very precise process that is only beginning to be understood. In Drosophila, during embryonic dorsal closure, the epithelial cells push the amnioserosa cells, which contract and eventually invaginate in the body cavity. In contrast, during pupariation the imaginal cells crawl over the passive larval tissue following a very accurate temporal and spatial pattern. Spreading is driven by filopodia and actin bridges that, protruding from the leading edge, mediate the stretching of the imaginal epithelia. Although interfering with JNK (Jun N-terminal kinase) and dpp produces similar phenotypic effects suppressing closure, their effects at the cellular level are different. The loss of JNK activity alters the adhesion properties of larval cells and leads to the detachment of the imaginal and larval tissues. The absence of dpp signaling affects the actin cytoskeleton, blocks the emission of filopodia, and promotes the collapse of the leading edge of the imaginal tissues. Interestingly, these effects are very similar to those observed after interfering with JNK and dpp signaling during embryonic dorsal closure (Martin-Blanco, 2000).

During larval development, the expression of puckered (puc) becomes detectable in larval tissues and some imaginal cells and the stalk and the region peripheral to the peripodial membrane of the wing, haltere, and leg discs. puc (monitored in the LacZ insertion puc^E69 heterozygous flies) is expressed in the leading edges of all thoracic discs (wing, halteres, and legs) and its expression is maintained during the spreading and fusion of the imaginal epithelium. Thus, puc colocalizes with the cells, leading the process of spreading and fusion in a manner that is comparable to dorsal closure in the embryo (Martin-Blanco, 2000).

Disc stalk opening and notum and wing blade eversion initiate about 3.5 h after puparium formation (APF). Once eversion is completed, it is possible to distinguish three phases leading up to the fusion of the wing imaginal discs. (1) The disc epithelium initiates its spreading toward the dorsal midline led by the most anterior puc-expressing cells. These cells will be the first to meet their contralateral counterparts at 5.5 h APF. (2) Immediately afterward, the most posterior imaginal cells spread, reaching the midline and fusing around 6 h APF. (3) Finally, centrally located cells close the gap at 6.5 h APF. At about that time, notum cells start to secrete their adult cuticle (Martin-Blanco, 2000).

Embryonic dorsal closure proceeds through the planar stretching of epidermal cells. Meanwhile, amnioserosa cells elongate in the apical-basal axis, invaginating only at the end of closure. In contrast, in pupae, imaginal cells roll over the larval tissue on their way to the dorsal midline, leaving behind several rows of larval cells. These larval cells delaminate from the edges of the larval epidermal sheet as imaginal cells proceed and undergo apoptosis (Martin-Blanco, 2000).

During embryonic dorsal closure, filamentous actin and nonmuscle myosin accumulate at the leading edge of the lateral epidermis and form a mechanically contiguous contractile band, or purse string. To evaluate the role of the cytoskeleton in thorax closure, the presence of actin was monitored. At early stages, imaginal cells contact across and over the larval epidermis, emitting filopodia. These filopodia form linear and branched structures that seem to originate in the imaginal epithelial edges and contact the imaginal cells of contralateral discs. Later, once the anterior ends of the discs have been brought together, filopodia and actin bridges are evident at posterior positions. At these stages, the cells of the leading edge change shape and undergo a prominent elongation toward the midline. This contradicts previous reports, which describe only round, polygonal cells and an accumulation of actin in the central midline upon fusion. Changes of shape align with the filamentous bridges, suggesting a mechanical role for these actin-rich structures (Martin-Blanco, 2000).

The JNK signaling cascade participates in the spreading and fusion of discs during pupation. Wing discs of maternally rescued homozygous hep^r75 (D-JNKK) animals remain in their initial position in the prepupa: they do not spread, and in many cases disc eversion does not take place. Milder defects can be observed in several other conditions in which JNK signaling is impaired. Thus, in hypomorphic heteroallelic combinations of kayak (a gene coding for the Drosophila homolog of the transcription factor c-Fos) or hep, the thoracic epithelia fail to reach the midline and fuse. Similar abnormalities are observed after overexpressing Puc or dominant-negative Fos with a Pnr-Gal4, a line that is expressed in the larval tissue and the central notum region ('pannier domain of expression') (Martin-Blanco, 2000).

To analyze the role of JNK signaling in leading-edge cells, the MZ980-Gal4 line was used. This Gal4 line is expressed specifically in the presumptive edge cells of wing and haltere discs and in the intervening larval cells. Expressing Puc ectopically with MZ980-Gal4 results in adults with a mild thorax cleft phenotype reminiscent of hep hypomorphic alleles (hep¹) (Martin-Blanco, 2000).

Complete failure of JNK signaling (in hep^r75 animals) abolishes both spreading and fusion. In many zygotic null animals thorax closure does not proceed and wings occasionally fail to evert. The expression of actin in hep^r75 animals is down-regulated in both the larval and the epidermal tissues. In these mutants, filopodia departing from leading-edge cells are rare or missing and imaginal cells do not change shape. In addition, the larval epidermal cells detach from each other and from the imaginal epidermis, round up, and their nuclei constrict, breaking up the integrity of the larval epithelium. As a consequence, the imaginal discs never spread and fuse (Martin-Blanco, 2000).

Imaginal disc epithelia have the general characteristics of other epithelia in Drosophila and in other organisms. The discs have a basal surface lined with a fibrous basal lamina and an apical surface at which cells are connected at their ends by a series of specialized junctions, including zonula adherens, gap, and septate junctions. Before eversion, the squamous cells of the peripodial epithelium are folded and adhere to the basal lamina. Just before eversion takes place, the cells detach from the basal lamina; the epithelial cells then columnarize and the accompanying contraction forces the discs to evert through the peripodial stalks. Stalk widening and disc eversion appear to result from microfilament contraction, which leads to dramatic changes in cell shape, rather than from changes in membrane adhesiveness (Martin-Blanco, 2000).

There are some differences between dorsal closure and imaginal spreading. During embryonic closure, the amnioserosa and the epidermal cells keep their relative positions constant, and despite occasional delaminations, amnioserosa cells remain in place until the very end of the process. They detach from the overlying epidermis only upon closure completion, become dispersed into the body cavity, and undergo apoptosis. By contrast, during disc spreading, imaginal cells crawl over the larval epidermis. In this process, larval cells are left below and behind and eventually delaminate from the edges (Martin-Blanco, 2000).

Spreading and fusion of epidermal cells could be directed by different mechanisms. In adult vertebrates, cells at the edge of cutaneous wounds extend lamellipodia and drag themselves forward. In contrast, in vertebrate embryos, wound-edge cells remain blunt-faced and the force to draw the wound edges together seems to be provided by a purse-string-like contraction of a thick cable of actin at the leading edge. This mechanism also applies to some developmental processes, such as Xenopus gastrulation and the late stages of C. elegans ventral enclosure. In Drosophila, purse-string contraction appears to be the mechanical force leading embryonic dorsal closure. Actin and non-muscle myosin accumulate at the leading edge of the epithelium; mutations in hep or in zipper (the gene coding for non-muscle myosin) that abolish actin and non-muscle myosin accumulation yield dorsal-open phenotypes (Martin-Blanco, 2000).

These data suggest that the spreading of the imaginal epithelium is active and led by cells at the boundary, although a contribution of the rest of the cells of the epithelia may be possible. Forward locomotion of imaginal cells probably will involve contraction of intracellular actomyosin filaments. Thick filopodia connecting cells to the contralateral heminota are also observed. These multibranched filopodia, which protrude out of leading-edge cells, expand over the larval surface and eventually form actin bridges. Upon contact, they seem to exert a mechanical force, pulling the imaginal tissues together.

The mechanical role of thick filopodia involved in imaginal spreading is conserved in other developmental processes such as gastrulation in the sea urchin embryo and the epiboly of the C. elegans hypodermis. In sea urchin, primary and secondary mesenchyme cells extend filopodia as they move, making contacts with the ectoderm. During ventral enclosure in the nematode, leading cells display actin-rich filopodia; treatment with cytochalasin D immediately halts the process (Martin-Blanco, 2000).

One important characteristic of epithelial fusion in a developmental context is the precise recognition of the contralateral parts. During embryonic dorsal closure in Drosophila, a perfect match links the anterior and posterior compartments of each segment across the midline. During pupariation, the spreading of the imaginal tissues results in the alignment of notal landmarks along the anterior-posterior axis. When rare mismatches occur, anterior cells never match to posterior ones; rather, they meet anterior cells of distinct contralateral segments. This accurate identification suggests that different positional values must be present in different cells at the leading edge and, importantly, a mechanism should exist that allows perception of these differences at a distance (Martin-Blanco, 2000).

Thorax closure starts at the anterior end of the wing disc, proceeds through the most posterior region, and, finally, fills the gap. This regulated cadence also has been observed in an independent study. Timing also is regulated during embryonic dorsal closure. In this process, spreading and fusion proceed from both ends of the embryo, showing segmental periodicity (Martin-Blanco, 2000).

Contact guidance is a mechanism that directs migration or spreading; in discs, contact guidance could be mediated by filopodial tracts making appropriate informative contacts at the contralateral discs. This situation would be reminiscent of sea urchin gastrulation, where thin filopodia are involved in cell-cell signaling. This function also has been suggested for cytonemes, actin-rich, thin filopodia present on Drosophila imaginal discs (Martin-Blanco, 2000).

In addition to morphological similarities, genetic evidence points to a similar molecular mechanism, led by the JNK signaling cascade, directing embryonic dorsal and imaginal thorax closure. During imaginal closure, JNK signaling affects the adhesion between imaginal and larval epidermal cells. In mutant conditions for this pathway, larval cells detach and prematurely degenerate, disrupting the continuity of the epithelium, which appears to be necessary for spreading and fusion. This defect also has been observed during embryonic dorsal closure. In embryos, after loss of both maternal and zygotic hep functions, amnioserosa cells detach from each other and from the epidermal cells and undergo premature cell death. Thus, JNK signaling appears to affect both the cytoskeleton and cell adhesiveness (Martin-Blanco, 2000).

The activity of JNK signaling is manifested by the expression of puc, which is detected in imaginal leading-edge cells and in a subset of cells of the peripodial membrane that ultimately will adopt a border position. Thus, the JNK pathway appears to determine the leading cells in the moving epithelia and to set up the borders between columnar and squamous epithelia. Alternatively, puc expression (and JNK signaling) could just reflect a particular physiological state of border cells (Martin-Blanco, 2000).

The JNK and Wg signaling pathways have been thought to function in distinct domains, with JNK regulating dorsal closure and Wg regulating segment polarity. In this study it has been demonstrated that Wg signaling is critical for normal dorsal closure and that a negative regulator of the JNK pathway, Puc, plays an unexpected role in ventral patterning. This connection emerged from the observation that reduction in Puc function suppresses both the dorsal closure and ventral segment polarity phenotypes of non-null mutations in armadillo mutants, which exhibit dorsal closure defects. Mutations in puckered, known from previous work to antagonize Jun N-terminal kinase in dorsal closure, also suppress, in a dose-sensitive manner, both the dorsal and ventral armadillo cuticle defects during segmentation. Activation of the Jun N-terminal kinase signaling pathway suppresses armadillo-associated defects in segmentation. Jun N-terminal kinase signaling promotes dorsal closure, in part, by regulating decapentaplegic expression in the dorsal epidermis. Wingless signaling is also required to activate decapentaplegic expression and to coordinate cell shape changes during dorsal closure. Together, these results demonstrate that MAP-Kinase and Wingless signaling cooperate in both the dorsal and ventral epidermis, and suggest that Wingless may activate both the Wingless and the Jun N-terminal kinase signaling cascades (McEwen, 2000).

The simplest hypothesis to explain these results is that puc suppresses arm by hyperactivating the JNK pathway. Consistent with this, the puc enhancer trap, a JNK target gene, is ectopically activated in certain ventral epidermal cells in puc mutants. In addition, activation of JNK signaling suppresses arm in a fashion very similar to that resulting from reduction in Puc function, and activation of a JNKKK in a wild-type embryo mimics weak activation of the Wg pathway (McEwen, 2000).

Zygotic loss-of-function mutations in the JNK pathway fail to appreciably affect ventral segment polarity, however. This is reminiscent of the role of JNK signaling in planar polarity. Loss-of-function mutations in the JNK pathway suppresses dominant activation of Fz or Dsh, but these mutations fail to exhibit planar polarity defects themselves. This may result from functional redundancy and/or cross-talk between different MAPKKs and/or MAPKs. Such crosstalk occurs: both DMKK4 and Hemipterous can activate Basket, while Drosophila p38 orthologs can phosphorylate Djun and ATF2, both known targets of Bsk. Thus, the JNK signaling pathway may function redundantly with other MAPK pathways, both in planar polarity and in segment polarity. As JNK-independent expression of puc has also reported, additional studies will be required to assess the ability of Puc to antagonize other MAPK signaling pathways. While these circumstantial arguments are consistent with a role for the JNK pathway in ventral patterning, the caveats raised by the lack of effects of loss-of-function JNK mutations leave open the possibility that Puc has a role in ventral patterning that is independent of its role in regulating JNK activity - for example, it could directly regulate the canonical Wg pathway (McEwen, 2000).

These data, combined with previous studies of JNK signaling, further suggest that Wg and JNK signaling act in parallel during dorsal closure. Both pathways regulate dpp expression in dorsal epidermal cells and are required for the proper coordinated cell shape changes to occur. These data are compatible with several different models. It may be that the two pathways both impinge on the same process and the same target gene, but that they do so in response to independent upstream inputs. However a potential direct connection between the Wg and JNK pathways is suggested. Using both genetics and in vitro studies, it has been demonstrated that JNK pathway kinases act downstream of Frizzled and Dsh in planar polarity and that Dsh can activate the JNK signaling cascade directly. This suggests that Dsh may function as a binary switch, deciding between the canonical Wg pathway and the JNK pathway during the establishment of segment polarity and planar polarity, respectively. Both the canonical Wg and the JNK pathways are required for proper dorsal closure, and both pathways affect expression of the same target gene, dpp. One plausible model accommodating these data is that Wg, acting via Frizzled receptors and Dsh, activates both the JNK pathway and the canonical Wg pathway simultaneously and in parallel during both dorsal closure and ventral patterning. The possibility that Wg activates both pathways, while exciting in principle, remains quite speculative, and must now be tested by more direct biochemical and cell biological means (McEwen, 2000).

It also is possible that Wg functions as a permissive signal required to allow other effectors to promote dpp expression. For example, dTCF (Pangolin) could repress dpp expression in the absence of Wg signaling by recruiting Groucho, a transcriptional repressor, to the dpp promotor. Wg signaling might relieve this repression by displacing Groucho with stabilized Arm. Consistent with this hypothesis, constitutive activation of Arm fails to rescue the dorsal closure defects of kayak/Fos-related antigen mutants. Thus activation of the canonical Wg signaling pathway is necessary but not sufficient to promote dpp expression. Wg signaling may thus only amplify JNK-dependent expression of dpp in the dorsal epidermis. One possible intersection between MAPK signaling cascades and TCF-mediated repression has been reported. Transcriptional repression of Wnt target genes in C. elegans depends upon POP-1, a TCF family member. POP-1 repressor activity is regulated by Mom-4, a Tak1-like kinase, and Lit-1, a Nemo-like MAP kinase relative (Nlk). In mammalian cells, the transcriptional activity and DNA-binding properties of TCF can be repressed by Tak1/Nlk activation. Therefore, the canonical Wg and MAPK/JNK pathways might converge at dTCF, with MAPK kinase signaling affecting dTCF activity. Additional studies will be required to assess the mechanism by which these pathways interact (McEwen, 2000).

The current model suggests that a sequential series of cellular events drive dorsal closure. Leading edge cells are thought to initiate closure by elongating in the DV axis and upregulating Dpp, thus signaling lateral cells to initiate similar cell shape changes. The events of dorsal closure apparently do not proceed in lockstep, with each event requiring the successful completion of the previous event. The stereotypical cell shape changes are lost in wg mutants; however, the lateral epidermal sheets usually meet at the dorsal midline. In contrast, while cell shape changes are initiated in arm mutants, though not in a coordinated fashion, the epidermis does not close. Further, as dpp expression in leading edge cells is lost in wg mutants, Dpp may not be essential for dorsal closure. Finally, because dorsal closure is more normal in wg than in JNK pathway mutants, the JNK pathway likely depends upon activation by signals other than Wg and must affect other processes in addition to Dpp signaling. Further work is required to clarify the semi-redundant mechanisms regulating dorsal closure (McEwen, 2000).

The phenotypic similarities between slipper and genes encoding the JNK signaling cascade, hep, bsk, and dJun, suggest that slpr may regulate JNK signaling. To further test whether slpr mutants diminish signaling through the JNK pathway, genetic epistasis tests were performed. Activation of positive components functioning downstream of slpr may be expected to alleviate the defect caused by slpr loss-of-function. Inducible expression of a constitutive active form of the Jun transcription factor that normally serves as a substrate for phosphorylation by Bsk significantly rescues the slpr mutant phenotype. Similarly, loss-of-function mutations in downstream negative components may augment residual signaling activity to functional levels. Consistent with this line of reasoning, slpr is dominantly suppressed by reducing the dosage of a negative regulator of JNK signaling, puc, encoding a JNK phosphatase. Heterozygosity at the puc locus significantly suppresses the severe cuticle phenotype of the strong slpr⁹²¹ allele, indicated by the clear reduction in size of dorsal cuticle holes. Moreover, loss of one copy of puc rescues embryos mutant for the weaker slpr^3P5 allele such that they develop to adulthood. Mutant male flies emerge but are weakly viable and show no gross morphological defects. Taken together, these data support a role for slpr in JNK signal transduction, upstream of bsk (Stronach, 2002).

Much of what is known about apoptosis in human cells stems from pioneering genetic studies in the nematode C. elegans. However, one important way in which the regulation of mammalian cell death appears to differ from that of its nematode counterpart is in the employment of TNF and TNF receptor superfamilies. No members of these families are present in C. elegans, yet TNF factors play prominent roles in mammalian development and disease. The cloning and characterization of Eiger, a unique TNF homolog in Drosophila, is described. Like a subset of mammalian TNF proteins, Eiger is a potent inducer of apoptosis. Unlike its mammalian counterparts, however, the apoptotic effect of Eiger does not require the activity of the caspase-8 homolog DREDD, but it completely depends on its ability to activate the JNK pathway. Eiger-induced cell death requires the caspase-9 homolog DRONC and the Apaf-1 homolog DARK. These results suggest that primordial members of the TNF superfamily can induce cell death indirectly by triggering JNK signaling, which, in turn, causes activation of the apoptosome. A direct mode of action via the apical FADD/caspase-8 pathway may have been coopted by some TNF signaling systems only at subsequent stages of evolution (Moreno, 2002).

Another pathway activated by mammalian TNF death factors is the JNK pathway, although its role in inducing apoptosis upon TNF signaling is less well defined. JNK signaling in Drosophila is reflected by the expression levels of puckered (puc), a gene encoding a dual-specificity phosphatase that forms a negative feedback loop by downregulating the activity of JNK. High levels of puc expression are induced by Eiger. Due to its function as a negative regulator of JNK, Puc can also be used as a powerful means to repress JNK activity if overexpressed by a constitutive promoter that is no longer dependent on this activity. Coexpression of Eiger and Puc completely blocks Eiger activity, strikingly reverting the eye and wing phenotypes to wild-type and blocking Eiger-induced elimination of cell clones. Forced expression of Puc does not prevent all forms of cell death. For example, when tested in the situation of polyglutamine repeat-induced neurodegeneration, which is also caused by apoptosis, coexpression of puc has no discernible protective effect. Taken together, these results are interpreted as firm evidence that Eiger induces the JNK signaling pathway and that Eiger-induced apoptosis is critically dependent on JNK activity (Moreno, 2002).

Consistent with this interpretation, several components of the Drosophila JNK pathway are rate limiting in mediating or preventing Eiger-induced apoptosis. The removal of one wild-type copy of either DTRAF1 (encoding the homolog of human TRAF2), misshapen (encoding a Ste20 kinase that binds to DTRAF1), or basket (encodes Drosophila JNK) suppresses Eiger-induced apoptosis. Conversely, animals heterozygous for a mutation in puc display an enhanced phenotype (Moreno, 2002).

The mechanism by which JNK signaling triggers cell death in response to TNF is poorly understood in mammals and is unknown in Drosophila. It was therefore of interest to identify the apoptotic machinery responsible for Eiger-induced cell death. Having excluded the caspase-8-like FADD/DREDD branch, focus was placed on the involvement of caspase-9, which represents another major pathway that leads to apoptosis. The key event for caspase-9 activation is its association with the protein cofactor Apaf-1 to form an active complex referred to as the apoptosome. Since many cell intrinsic insults can trigger this pathway, it has been termed the 'intrinsic death pathway'. Expression of a dominant-negative form of the Drosophila caspase-9 homolog DRONC, comprising only the CARD domain, fully blocks Eiger-induced apoptosis in a dose-dependent manner. Moreover, genetic removal of DARK, the homolog of Apaf-1, suppresses Eiger-dependent phenotypes. These results indicate that the presumptive Drosophila apoptosome is essential for the ability of Eiger to induce cell death. In agreement with this conclusion, overexpression of Thread, the Drosophila inhibitor of apoptosis protein 1 (DIAP1) blocks Eiger function. Thread/DIAP1 has been shown to bind DRONC and target it for degradation. Most instances of programmed cell death that have been analyzed in Drosophila are triggered by, and require, the genes reaper, hid, or grim, which encode small proteins that bind to and inactivate IAPs, such as Thread/DIAP1. The removal of one copy of a chromsosomal segment that includes the genes hid, grim, and reaper rescues eye ablation, and Eiger induces a strong transcriptional activation of hid and a weak activation of reaper. These results suggest, therefore, that Eiger/JNK signaling triggers DRONC by inactivating the IAPs via a transcriptional upregulation of hid (Moreno, 2002).

Puckered and the immune response

Innate immunity is essential for metazoans to fight microbial infections. Genome-wide expression profiling was used to analyze the outcome of impairing specific signaling pathways after microbial challenge. These transcriptional patterns can be dissected into distinct groups. In addition to signaling through the Toll/NFkappaB or Imd/Relish pathways, signaling through the JNK and JAK/STAT pathways controls distinct subsets of targets induced by microbial agents. Each pathway shows a specific temporal pattern of activation and targets different functional groups, suggesting that innate immune responses are modular and recruit distinct physiological programs. In particular, the results may imply a close link between the control of tissue repair and antimicrobial processes (Boutros, 2002).

Lipopolysaccharides (LPS) are the principal cell wall components of gram-negative bacteria. In mammals, exposure to LPS causes septic shock through a Toll-like receptor TLR4-dependent signaling pathway. LPS treatment of Drosophila SL2 cells leads to rapid expression of antimicrobial peptides, such as Cecropins (Cec). SL2 cells resemble embryonic hemocytes and have also been used as a model system to study JNK and other signaling pathways. LPS-responsive induction of the antimicrobial peptides AttacinA (AttA), Diptericin (Dipt), and Cec relies on IKK and Relish. In order to obtain a broad overview on the transcriptional response to LPS in Drosophila, genome-wide expression profiles of SL2 cells were generated at different time points following LPS treatment. Altered expression of 238 genes was detected (Boutros, 2002).

In time-course experiments, a complex pattern of gene expression was observed that can be separated into different temporal clusters. A first group, with peak expression at 60 min after LPS, primarily consists of cytoskeletal regulators, signaling, and proapoptotic factors. This group includes cytoskeletal and cell adhesion modulators such as Matrix metalloprotease-1, WASp, Myosin, and Ninjurin, proapoptotic factors such as Reaper, and signaling proteins such as Puckered and VEGF-2. A second group, with peak expression at 120 min, includes many known defense and immunity genes, such as Cec, Mtk, and AttA, but not the gram-positive-induced peptide Drs. Interestingly, this cluster also includes PGRP-SA, which is a gram-positive pattern recognition receptor in vivo, suggesting possible crossregulation between gram-positive- and gram-negative-induced factors. A third group is transiently downregulated upon LPS stimulation. This cluster includes genes that play a role in cell cycle control, such as String and Rca1. Altogether, these results show that, in response to LPS, a defined gram-negative stimulus, cells elicit a complex transcriptional response (Boutros, 2002).

In the Rel-independent group, several transcripts were identified that are indicative of other signaling events. For example, puc is transcriptionally regulated by JNK signaling during embryonic development. Therefore, the effect of removing SAPK/JNK activity was tested on LPS-induced transcripts. mkk4/hep dsRNA-treated cells lose the ability to induce the Rel-independent cluster, indicating that LPS signaling branches downstream of Tak1 into separate Rel- and JNK-dependent branches. To validate the results obtained from the microarray experiments, quantitative PCR (qPCR) was performed using puc and cec mRNA levels as indicators for Imd/Rel- or Mkk4/Hep-dependent pathways. Additionally, the effect of removing imd, which, in vivo, acts upstream of Tak1, was tested to clarify whether, in addition to Tak1, other known upstream components of a gram-negative signaling pathway are required for both Rel- and Mkk4/Hep-dependent pathways. These qPCR experiments confirm that cec is dependent for its expression on Imd, Tak1, Rel, and Key, whereas LPS-induced puc expression is dependent on Imd, Tak1, and Mkk4/Hep. Hence, the immunity signaling pathway in response to LPS bifurcates downstream of Imd and Tak1 into Rel- and SAPK/JNK-dependent branches. Both the Rel and SAPK/JNK pathways regulate different functional groups of downstream target genes (Boutros, 2002).

While both Rel and Mkk4/Hep pathways are downstream of Imd and Tak1 in response to LPS, the two downstream branches elicit different temporal expression patterns. It was then asked whether the first transcriptional response is controlled by downstream targets that might negatively feed back into the signaling circuit. puc was a candidate for such a transcriptionally induced negative regulator. Expression profiles of cells depleted for puc were tested before and after a 60 min LPS treatment. These experiments showed that transcripts dependent on the Mkk4/Hep branch of LPS signaling are upregulated, even without further LPS stimulus. In contrast, Rel branch targets are not influenced. puc dsRNA-treated cells show loss of the typical round cell shape. These cells appear flat and have a delocalized Actin staining, consistent with a deregulation of cytoskeletal modulators in puc-deficient cells (Boutros, 2002).

The analysis of expression profiles shows that, while SAPK/JNK and Rel signaling are controlled by the same Imd/Tak1 cascade, they appear to have different feedback loops. Whereas Rel signaling induces Rel expression and thereby generates a self-sustaining loop, possibly leading to the maintenance of target gene expression, the SAPK/JNK branch induces an inhibitor and thereby establishes a self-correcting feedback loop. These results may explain how a single upstream cascade can lead to different dynamic patterns (Boutros, 2002).

Since the expression of cytoskeletal genes after LPS stimulation is dependent on a JNK cascade, whether removing JNK activity in vivo affects the induction of fln was examined. In Drosophila, JNK signaling pathways have been previously implicated in epithelial sheet movements during embryonic and pupal development, a process that has been likened to wound-healing responses. hep¹ (JNKK) mutants, which are impaired in JNK signaling, the induction of fln is diminished, whereas the expression of the antimicrobial peptide dipt is not affected. A test was performed to see whether fln induction in Tl loss-of-function alleles is affected. These experiments show that fln expression is lost in Tl mutants, suggesting that Toll acts upstream of a JNK pathway to induce septic injury-induced target genes (Boutros, 2002).

NFkappaB pathways play a central role for innate and adaptive immune response in mammals. In innate immune responses, TLRs on dendritic cells recognize microbial agents and activate NFkappaB, leading to the expression of proinflammatory cytokines and other costimulatory factors required to initiate an adaptive immune response. Additionally, other signaling pathways have been implicated at later stages during immune responses in mammals, but their physiological role in innate immunity remains rather poorly understood. For example, several cytokines, such as IL-6 and IL-11, signal through a JAK/STAT pathway to induce the expression of acute phase proteins. Similarly, JNK pathways are activated in response to TNF and IL-1, may lead to the expression of immune modulators, and are required for T cell differentiation. In Drosophila, studies have investigated two distinct NFkappaB-pathways --Toll and Imd/Rel -- that have been shown to mediate gram-positive/fungal and gram-negative responses. Both pathways induce specific antimicrobial peptides and thereby focus the response on the invading microbial agent. Genetic analysis has shown that functions of the NFkappaB-pathways are separable; flies that are mutant for only one of these pathways are susceptible to subgroups of pathogens. Could the contribution of NFkappaB-dependent and, possibly, other signaling pathways be identified by examining global expression profiles? The obtained data set demonstrates that NFkappaB-independent signaling pathways contribute to the transcriptional patterns observed after microbial infection. Both in cells and in vivo, JNK-dependent targets precede the peak expression of antimicrobial peptides that require NFkappaB. JAK/STAT targets are induced with a distinct temporal pattern that shows late, but only transient, expression characteristics. The stereotyped pathway patterns after microbial challenge suggest that the correct temporal execution of signaling events, similar to signaling during development, may play an important role in the regulation of homeostasis (Boutros, 2002).

Strikingly, cytoskeletal gene expression during innate immune responses is controlled by JNK through the same upstream signaling cascade that activates NFkappaB pathways. JNK pathways act downstream of microbial stimuli, both in vivo and in cells, to induce cytoskeletal regulators. In SL2 cells, JNK signaling is required for the induction of a cluster of cytoskeletal, cell adhesion regulators and proapoptotic factors. Interestingly, both NFkappaB and JNK branches share the same upstream components, Tak1 and Imd, indicating that the activation of both processes are tightly linked. MMP-1, a matrix metalloproteinase that is one of the most markedly upregulated genes after LPS stimulation, has been implicated in wound-healing responses in mammals. Compared with experiments in cells, the situation in vivo after septic injury is likely more complex. Gene expression profiling in whole organisms likely has a lower sensitivity for transcriptional changes that occur in rather small numbers of cells. Also, tissue-specific differences in signaling pathway activity may not reflect the transcriptional changes observed in the cell culture model. Muscle-specific cytoskeletal factors, possibly because they were injected into the thoracic muscle, are not inducible in a JNK-deficient genetic background. However, since it was necessary to remove both Mkk4 and Hep (Mkk7) in cells to deplete JNK pathway activity, an experiment that cannot be performed in vivo because of the lack of an Mkk4 mutant, these experiments might not have uncovered all JNK-dependent transcripts. SAPK/JNK modules can also be linked to different upstream activating cascades. For example, a recent study reported the activation of p38a through a cascade involving Toll, TRAF6, and TAB. Similarly, during innate immune responses JNK pathways can be activated by both Toll and Imd pathways in vivo (Boutros, 2002).

The activation of JNK signaling is reminiscent of signaling during dorsal and thorax closure. In dorsal closure, SAPK/JNK signaling controls cytoskeletal rearrangements that lead to the epithelial sheet movements of the embryonic epidermis. SAGE analysis of embryos with activated SAPK/JNK signaling has shown an induction of cytoskeletal factors. Also, dorsal closure movements are proposed to be similar to the reepithelization that occurs during wound healing. In other developmental contexts, SAPK/JNK signaling has been implicated in cytoskeletal rearrangements and cell motility, such as the generation of planar polarity in Drosophila and convergent-extension movements in vertebrates. A common theme of SAPK/JNK pathways might be their control of cytoskeletal regulators for diverse biological processes. The finding that, in response to LPS, SAPK/JNK and NFkappaB targets are coregulated through the same intracellular pathway suggests a close linkage of directed antimicrobial activities and tissue repair processes (Boutros, 2002).

In conclusion, genome-wide expression profiling was employed to examine the contribution of different signaling pathways in complex tissues and to assign targets to candidate pathways. Both a cell culture model system and an in vivo analysis were used to show the temporal order of NFkappaB-dependent and -independent pathways after septic injury. An interesting question that remains is, how do the extracellular events leading to pathway activation reflect the nature of the pathogen? Clean injury experiments induce a largely overlapping set of induced genes, but to a lower extent than septic injury. This is consistent with experiments showing that septic injury with only gram-negative E. coli induces both anti-gram-negative and anti-gram-positive responses. These results can be interpreted to suggest that wounding, in itself, might be sufficient to induce a transient (and unspecific) innate immune response. However, further studies are needed to understand the nature of the inducing agent (Boutros, 2002).

JNK signaling confers tolerance to oxidative stress and extends lifespan in Drosophila

Changes in the genetic makeup of an organism can extend lifespan significantly if they promote tolerance to environmental insults and thus prevent the general deterioration of cellular function that is associated with aging. This study introduces the Jun N-terminal kinase (JNK) signaling pathway as a genetic determinant of aging in Drosophila. Based on expression profiling experiments, it is demonstrated that JNK functions at the center of a signal transduction network that coordinates the induction of protective genes in response to oxidative challenge. JNK signaling activity thus alleviates the toxic effects of reactive oxygen species (ROS). In addition, flies with mutations that augment JNK signaling accumulate less oxidative damage and live dramatically longer than wild-type flies. This work thus identifies the evolutionarily conserved JNK signaling pathway as a major genetic factor in the control of longevity (Wang, 2003).

JNK phosphorylates a variety of transcription factors and enhances their transcriptional activation potential. Thus, insight into the biological consequences of stress-activated JNK signaling might be gained by analyzing the relevant downstream genetic programs. Drosophila was chosen as a model organism for such studies, since its JNK pathway is genetically very tractable. The multiplicity of homologous mammalian kinases that are functionally, at least partially, redundant (three JNK genes produce at least ten protein isoforms), has impeded similar analyses in mammals. The genomic response to JNK signaling has been mapped in the Drosophila embryo using serial analysis of gene expression. Among the genes induced in embryos with increased JNK signaling, a group was identified with tentative functions in cellular stress responses as well as several genes that are known to be activated in response to oxidative damage. In an independent experiment, similar genes were found to be upregulated in response to JNK signaling in differentiating photoreceptors. These findings suggested that JNK signaling activates a gene expression program that confers tolerance to oxidative stress in a variety of cell types. To test this hypothesis, the expression was monitored, in the adult fly using quantitative real-time RT-PCR, of four representative genes (hsp68, gstD1, fer1HCH, and mtnA), which were identified as JNK dependent in the SAGE experiments. The induction of the respective mRNAs in response to oxidative stress, artificially brought on by treatment with the drug paraquat, was measured in wild-type flies and in hemizygotes for hep¹, a hypomorphic allele of the Drosophila JNKK gene, hemipterous (hep). Paraquat, a compound widely used to apply oxidative stress to cells and organisms, leads to continuous intracellular generation of O₂^.- radicals. It efficiently activates JNK in the fly, as indicated by the transcriptional activation of puckered (puc), one of the prototypical target genes of JNK signaling in Drosophila. puc encodes a JNK-specific phosphatase that downregulates the pathway, thus establishing a negative feedback loop. RT-PCR data show that JNK signaling is required for the induction of the four tested genes in response to oxidative stress, supporting the notion that flies react to oxidative challenge with a protective gene expression program dependent, at least in part, on JNK signal transduction (Wang, 2003).

To examine the relevance of JNK signaling for the sensitivity of the organism to oxidative stress, adult flies were exposed to paraquat for a prolonged period of time and their survival was monitored. Compared to wild-type animals, flies with decreased JNK signaling potential (hemizygotes for hep¹, or heterozygotes for a hypomorphic allele of the Drosophila JNK gene basket, bsk²) were more sensitive to moderate doses of paraquat. Conversely, flies gained resistance to paraquat when signal flow through the kinase cascade was promoted by overexpression of Bsk or Hep. Similarly, boosting JNK signal transduction by reducing the gene dose of puc, conferred strong paraquat resistance in a hep- and bsk-dependent fashion. Flies heterozygous for puc exhibit elevated levels of JNK activity, as inferred by the dosage sensitivity of JNK-mediated apoptotic phenotypes in the developing wing, as well as by rescue of developmental defects normally observed in flies carrying hep and kay mutations. Constitutive overexpression of one of the identified JNK-inducible stress response genes, Hsp68, also protects flies against oxidative stress, suggesting that JNK's downstream genetic program mediates the observed protection. The observed differences in sensitivity to paraquat were not due to feeding abnormalities or a general tolerance to toxic compounds of the tested genotypes, since they are similarly sensitive to G418 toxicity (Wang, 2003).

Tissue-specific overexpression of superoxide dismutase (SOD) in motorneurons increases the resistance to oxidative stress and extends the lifespan of Drosophila. This result suggests neurons as the 'weakest link' in the organism's tolerance to oxidative insults and as a cell type in which protective mechanisms would be most critical. To investigate whether JNK signaling in neurons could play a role in such mechanisms, fly strains were examined in which Hep overexpression was directed either to the nervous system or to muscle tissue in an RU486-inducible manner (using the 'gene-switch Gal4' driver). The toxicity of paraquat was reduced significantly when Hep was expressed in the nervous system (ELAV Gal4 drives expression in all cells of the peripheral and central nervous systems but not when it was expressed in the musculature). This result highlights a specific function of JNK signaling in the protection of neurons against oxidative stress. While it cannot be ruled out that JNK may also act protective in nonneuronal cells (for instance in muscle cells), such protection seems not to be sufficient for the organism's survival, indicating that protection of neurons is critical (Wang, 2003).

Importantly, the inducibility of the JNK effect by RU486 in this system rules out variations in the genetic background as an explanation for differences in paraquat sensitivity (Wang, 2003).

According to the free radical theory of aging, one genetic determinant for the lifespan of an organism is its sensitivity to oxidative stress. It was asked whether the protection against oxidative damage that is brought about by an increase in JNK signaling potential might be sufficient to extend Drosophila's life expectancy. Flies heterozygous for puc were examined to test this hypothesis, since the experiments demonstrated that the tolerance of flies to oxidative stress increases with decreasing gene dose of puc. Flies heterozygous for either one of two different loss-of-function alleles of puc (puc^A251.1 or puc^E69) showed dramatic extensions of median and maximum life expectancy compared to wild-type flies and to flies of an isogenic control strain. The difference in the degree of lifespan extension by the two alleles correlates well with their described allelic strength. The results thus suggest a direct relationship between the decrease of Puc activity in the mutants and the resulting lifespan extension. Since biochemical and genetic data indicate that the activity of Puc is limited to the JNK signaling pathway (as opposed to other MAPK pathways, the lifespan extension in puc mutants is likely to be caused by higher levels of JNK signaling. The requirement for a functional JNK pathway in the longevity of puc mutants was tested directly by comparing the lifespan of puc^E69 heterozygous males in a wild-type background to puc^E69 heterozygotes in a hep¹ hemizygous backgound. Heterozygosity for puc leads to an only modest increase in mean and maximum lifespan of hep¹ hemizygous flies, indicating that a functional JNK cascade is required for efficient lifespan extension in puc mutants. These results strongly support the notion that the longevity phenotype observed in puc mutants is due to an increase in JNK signaling activity (Wang, 2003).

The genomic experiments suggested that elevated JNK signaling activity causes higher basal levels of protective genes. Whether constitutive overexpression of one of the identified JNK target genes, hsp68, would be sufficient to extend lifespan of Drosophila was tested. In agreement with the hypothesis, small but significant increases were observed in mean and maximum lifespan in flies that overexpress hsp68 compared to isogenic wild-type controls. This experiment is consistent with observations that increased expression of chaperones extend the lifespan of Drosophila (Wang, 2003).

Providing higher JNK signaling levels in neuronal tissue is sufficient to increase oxidative stress tolerance. To test whether neuronal-specific protection would also be sufficient to extend lifespan of the organism, survival was monitored of flies that overexpress Hep constitutively in neuronal tissue under the control of ELAV Gal4. Neuronal overexpression of Hep extended lifespan significantly, indicating that the level of JNK activity in neuronal tissue determines not only the fly's oxidative stress tolerance, but also its lifespan. Importantly, these results confirm, independently of puc mutations, that JNK signaling promotes longevity (Wang, 2003).

Several genetically determined changes in physiology have been associated with extended lifespan in Drosophila. Such changes include reduced reproductive activity, dwarfism, delays in development, as well as stress tolerance. Whether the JNK pathway might affect parameters indicative of such physiological changes was examined. puc heterozygotes and wild-type controls exhibit roughly equivalent sizes (as determined by body weight), reproductive activities (fecundity), as well as developmental timing. In contrast, oxidative stress tolerance and tolerance to starvation differ markedly between wild-type and puc heterozygous flies. Importantly, 10-day-old puc heterozygotes contain significantly decreased levels of oxidized proteins. The quantity of protein oxidation products, such as polypeptides carrying carbonyl groups, is a measure for the accumulated oxidative damage suffered by an organism. Taken together, these results suggest that increased JNK signaling is sufficient to reduce oxidative damage throughout the lifetime of a fly and that this beneficial effect may be the cause of the longevity phenotype of gain-of-function mutants for this signaling pathway (Wang, 2003).

This work identifies the JNK signaling pathway as a significant genetic determinant of longevity in Drosophila. Activation of JNK in response to oxidative challenge and to other environmental insults has been well described in a number of model systems and was proposed to trigger the expression of genes that could mediate protective functions on the organism at least in certain cell types. Against this backdrop, resulting in the prediction that JNK signaling would protect the organism from oxidative challenge, it may seem surprising that, until now, no evidence has been produced that links JNK signaling to an extended lifespan. Evidently, experimental limitations of mammalian systems, including increased functional complexity and genetic redundancy, have precluded clear-cut experiments to address this question (Wang, 2003).

While unidentified functions of JNK signaling that might be relevant to the aging process cannot be excluded, it seems plausible (and the free radical theory of aging would predict) that the observed protection against oxidative insults decisively delays aging and thus causes the longevity phenotype of puc heterozygotes. Earlier observations, as well as the current experiments, support this notion: Hsp70, and its JNK-inducible relative Hsp68, have been shown to extend lifespan when overexpressed in Drosophila. These chaperones have been implicated in oxidative stress resistance and may have repair functions downstream of JNK signaling. The reduced level of oxidative damage in aging puc heterozygotes further supports this view. JNK signaling thus emerges as an evolutionarily conserved gene-regulatory network that limits oxidative damage in the organism and its impact on aging (Wang, 2003).

Apoptotic cells can induce compensatory cell proliferation through the JNK and the Wingless signaling pathways

In many metazoans, damaged and potentially dangerous cells are rapidly eliminated by apoptosis. In Drosophila, this is often compensated for by extraproliferation of neighboring cells, which allows the organism to tolerate considerable cell death without compromising development and body size. Despite its importance, the mechanistic basis of such compensatory proliferation remains poorly understood. Apoptotic cells are shown to express the secretory factors Wingless and Decapentaplegic. When cells undergoing apoptosis were kept alive with the caspase inhibitor p35, excessive nonautonomous cell proliferation is observed. Significantly, Wg signaling is necessary and, at least in some cells, also sufficient for mitogenesis under these conditions. Finally, evidence is provided that the DIAP1 antagonists reaper and hid can activate the JNK pathway and that this pathway is required for inducing wg and cell proliferation. These findings support a model where apoptotic cells activate signaling cascades for compensatory proliferation (Ryoo, 2004).

To investigate how the inhibition of diap1 may lead to mitogen expression, attention was focused on Dronc and the Jun N-terminal Kinase (JNK) pathway. Dronc has been implicated in compensatory proliferation, and its activity can be inhibited by the expression of dronc^DN. In addition, the JNK signaling pathway was considered as a candidate, since its activity is known to correlate with many forms of stress-provoked apoptosis, including disruption of morphogens, cell competition, and rpr expression. In Drosophila, the JNK pathway can be effectively blocked by the expression of puckered (puc), which encodes a phosphatase that negatively regulates JNK (Ryoo, 2004).

To induce patches of undead cells, wing imaginal discs were generated with mosaic clones expressing hid and p35. 48 hr after induction, these imaginal discs contained hid-expressing clones that autonomously induced wg. Using this experimental setup, it was asked whether additional expression of either dronc^DN or puc would block wg induction in undead cells. When dronc^DN was coexpressed, a subset of the hid-expressing population was still able to induce wg. In contrast, when puc was coexpressed, wg induction by hid was almost completely blocked. These results provide evidence that the JNK pathway is required for wg induction under these conditions but fail to uncover a similar requirement for Dronc (Ryoo, 2004).

To independently investigate the role of puc and dronc^DN in compensatory proliferation, the size of wing discs harboring undead cells was measured and they were compared with those of the sibling controls. Under the experimental conditions, wing discs harboring hid- and p35-expressing clones were on average 53% larger than their sibling controls. Coexpression of puc within these undead clones significantly limited growth, resulting in only a small increase in wing disc size that was not statistically significant. In contrast, coexpression of dronc^DN did not limit growth. Wing size measurements also correlated with the degree of wg induction. The larger size of discs harboring hid- and p35-expressing cells is not due simply to extra cell survival: (1) these undead cells are derived from the normal lineage; (2) the size of wing discs expressing hid, p35, and puc serves as a control. In this case, although a large number of undead cells were generated, no significant increase in disc size was observed, in stark contrast to the discs expressing hid and p35 only. It is concluded that the JNK pathway is required for the nonautonomous growth promoting activity of the undead cells (Ryoo, 2004).

To confirm a role of puc in imaginal disc growth, rpr and p35 werecoexpressed in wild-type and puc^−/+ imaginal discs. Like hid, rpr is a DIAP1 antagonist, but with a weaker cell killing activity when overexpressed in imaginal disc cells. In a puc^+/+ background, a small amount of ectopic wg expression was observed, indicative of rpr's weaker DIAP1 inhibiting activity. In contrast, ectopic wg expression was strongly enhanced in puc^−/+ discs. Because the puc allele used, puc^E69, also acts as a lacZ reporter, JNK pathway induction could be monitored simultaneously. wg induction in undead cells correlates very well with puc-lacZ expression, with a stronger induction at the center of the wing pouch. These results further support the role of JNK in the induction of wg (Ryoo, 2004).

Next to be tested was whether the reduction of puc had an effect on apoptosis-induced cell proliferation. Whereas puc^−/+ discs expressing only p35 had BrdU incorporation similar to wild-type discs, coexpression of rpr and p35 in puc^−/+ led to a significant increase in BrdU incorporation. Also, the size of these discs were on average 41% larger than those coexpressing rpr and p35 in a puc^+/+ background. Taken together, these results show that diap1 inhibition leads to JNK activation and that JNK activity promotes wg induction and cell proliferation (Ryoo, 2004).

To directly test if JNK signaling can activate wg and dpp expression, hep^CA, a constitutively active form of hemipterous (hep), the Drosophila JNK kinase was conditionally expressed. Expression of hep^CA causes induction of wg-lacZ within 22 hr and to a lesser extent also dpp-lacZ. These ß-gal-expressing cells shifted basally and were apoptotic as assayed by anti-active caspase-3 antibody labeling. Hid protein levels were also elevated in these cells. Significantly, since p35 was not use to block apoptosis in this experiment, this demonstrates that wg and dpp can be induced not only in undead cells, but also in 'real' apoptotic cells (Ryoo, 2004).

This study provides evidence that the central apoptotic regulators can control the activity of mitogenic pathways. In particular, inhibition of DIAP1, either via expression of Reaper and Hid or by mutational inactivation, leads to the induction of the putative mitogens wg and dpp. When apoptosis was initiated through DIAP1 inhibition but cells were kept alive by blocking caspases, the resulting 'undead cells' exhibited strong mitogenic activity and stimulated tissue overgrowth. Inhibiting wg signaling with a conditional TCF^DN blocked cell proliferation in imaginal discs, indicating that wg has an essential mitogenic function. Finally, evidence was provided that the JNK pathway mediates mitogen expression and imaginal disc overgrowth in response to rpr and hid. Based on these results, it is proposed that apoptotic cells actively signal to induce compensatory proliferation. DIAP1 inhibits both caspases as well as dTRAF1. According to this model, when DIAP1 is inhibited in response to cellular injury, the JNK pathway is activated and wg/dpp are induced in apoptotic cells. Secretion of these factors stimulates growth of proliferation-competent neighboring cells and leads to compensatory proliferation (Ryoo, 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 of the processes that best exemplify the dramatic changes that shape organisms is insect metamorphosis. In Drosophila and other holometabolous insects, most of the larval structures are replaced with new tissues that will give rise to the adult or imago. In particular, the adult epidermis with the exception of the abdominal structures develops from imaginal epithelial discs. During larval stages, the primordia of imaginal discs, set during embryogenesis, invaginate and grow to become flattened sacs arranged in a monolayer epithelium connected to the larval epidermis by a stalk. The mature discs contain two populations of cells, a columnar epithelium that will give rise to most of the adult structures and a thinner and more squamous peripodial epithelium (PE) with a reduced contribution to adult tissues. Upon metamorphosis, the imaginal discs undergo striking morphological changes, everting, expanding, and fusing to ipsilateral and contralateral adjacent discs generating the adult exoskeleton (Pastor-Pareja, 2004).

The process of movement and sealing of imaginal discs and, in general, epithelial sheets can be subdivided into three sequential steps: (1) leading cells are specified and brought into position; (2) cells execute coordinated forward movements by changing shape and/or migrating over a substratum, and (3) sheets merge and fuse. Most recent work on disc morphogenesis has focused on the cellular and molecular events underlying their late expansion and fusion, while the mechanisms involved in disc eversion have been poorly explored in vivo (Pastor-Pareja, 2004).

In late third instar larvae, the steroid molting hormone 20-hydroxyecdysone is believed to coordinate the almost simultaneous eversion of all discs by inducing a contraction of the PE. This is thought to drive movement of the appendages to the outside of the larval epidermis through relaxed and widened disc stalks. This classical view is supported by in vitro studies showing that treatment of cultured discs with ecdysone is sufficient to induce eversion and that contraction of an intact PE is necessary to achieve this goal. These descriptive reports have led to the proposition that cell shape changes (longitudinal contraction in the PE and circumferential elongation at the disc stalks) are sufficient for imaginal disc eversion. However, there are as yet no data to confirm that this mechanism exists in vivo and no convincing explanation on how a stalk of no more than ten cells in diameter could achieve the width required to allow the entire disc (more than 60,000 cells) to pass through. Further, this accepted view neglects earlier proposals suggesting a different eversion mechanism mediated by the rupture of the PE. A model supported by fate maps has been developed for the PE of Calliphora, a related dipteran, imaginal wing discs (Pastor-Pareja, 2004).

Several studies have revealed a requirement for cytoskeletal components and a number of signal transduction molecules for imaginal disc morphogenesis during the first hours of metamorphosis. The latter include the Drosophila AP-1 transcription factors, D-Jun and D-Fos (Kayak [Kay]), and an upstream kinase cascade homologous to the Jun-NH2-terminal kinase (JNK) pathway in mammals. The core of this cascade is formed by the stress-activated kinases JNKK and JNK. In Drosophila, JNKK and JNK homologs are encoded by the genes hemipterous (hep) and basket (bsk). JNK signaling mutant larvae do not spread their discs in the process of thorax closure. This phenotype is accompanied by a loss of puckered (puc) expression in the disc stalk and the PE. Puc is a dual-specificity phosphatase that selectively inactivates Bsk and, thus, is thought to act in a negative feedback loop. JNK activity is necessary to maintain the adhesion of the imaginal leading edge cells to their larval substrate and to promote actin dynamics (lamellipodia and filopodia formation). It has been shown that this signaling cascade also regulates the process of embryonic dorsal closure, where the embryonic epidermis fuses along the dorsal midline. Based on these similarities, it has been suggested that a conserved mechanism regulates the spreading and fusion of epithelial sheets (Pastor-Pareja, 2004).

A new morphogenetic/cellular mechanism has been uncovered for disc eversion based on histological sections and direct observation of imaginal morphogenesis in vivo. At the onset of metamorphosis, imaginal discs coordinately appose their peripodial sides and stalks (PS cells) to the larval epidermis. Then, eversion proceeds through the progressive invasion of the larval epidermis by PS cells undergoing a pseudo-epithelial-mesenchymal transition (PEMT). Multiple perforations in the peripodial/larval bilayer are thus generated: these coalesce with the disc stalk into a single hole, widening the gap and allowing disc evagination. When eversion is complete, the PS cells localize to the leading front of the discs, spearheading their expansion over larval cells. The roles of the JNK pathway at discrete steps of disc morphogenesis progression have been analyzed. The JNK cascade functions to promote the apposition of PS and larval cells, to determine the degree of PEMT and the motility of leading edge/PS cells, and to maintain the adhesion between the larval and imaginal tissue. It is proposed that this molecular mechanism can be relevant to morphogenetic processes of perforation of transient epithelia in different phyla (Pastor-Pareja, 2004).

The current view of imaginal disc eversion asserts that the externalization of appendage primordia proceed through widened discs' stalks during early pupal development. However, a detailed analysis of PS cell markers appears to challenge this simple inversion mechanism (Pastor-Pareja, 2004).

In early third instar imaginal wing discs, the gene puc is expressed at high levels in stalk cells and some PE cells. This expression evolves through the third instar until all PS cells (about 700 in the mature wing disc) express puc at the white prepupa stage (0-1 hr hours after puparium formation [APF]). These dynamic changes of puc expression are also observed in leg, haltere, and eye discs. The PS expression of puc strictly depends on JNK activity, and it is abolished from mutant hep (JNKK) larvae or after Puc overexpression. Thus, a JNK signaling feedback loop, first described during embryonic dorsal closure, is shared by PS cells at the onset of the eversion and closure of the discs. During wing disc eversion, only cells found at the edge of the hole through which the disc everts and at the leading front mediating fusion to adjacent prothoracic, mesothoracic, and metathoracic discs show puc expression, and hence JNK activity. Importantly, marking all cells that have expressed puc as well as their descendants shows that puc-expressing cells do not change their identity, nor do they die or get excluded from the epithelium until the end of the disc fusion process, when most of these cells are lost. Hence, all PS cells are recruited to the front edge during disc eversion (Pastor-Pareja, 2004).

These findings lead to a topological dilemma. In order to reach their final position at the leading front, the PS cells would need to reposition themselves within the epithelia. Although this rearrangement just could be achieved through a massive constriction of the PE, a complementary mechanism has been uncovered, which involves larval epidermis perforation and PE cells intercalation (Pastor-Pareja, 2004).

At third instar larval stages, the wing disc obliquely hangs from the larval epidermis, which is separated from the peripodial surface of the disc at the notum level by several larval muscles and tracheal tubules. The disc and the larval epidermis are isolated by their corresponding extracellular basal lamina. During late third instar stages and the first hours APF, the notum-wing side of the disc folds progressively to acquire the adult organ shape. At the initiation of pupariation, the disc affixes to the larval epidermis through its peripodial side. At 3 hr APF, the PS cells lose their squamous shape to adopt a more rounded one and are found in close contact with the larval epidermal cells via their basal surfaces. Multiple actin-rich protrusions lead this apposition. At this step, the basal lamina in between both layers degrades, leading to an intimate adhesion (Pastor-Pareja, 2004).

Once imaginal discs appose the larval epidermis, PS cells, mostly around the disc stalk, invade the larval epithelium, gradually replacing the larval cells at the pupal surface without compromising the integrity of the peripodial sheet. Several holes are opened in the peripodial/larval bilayer, which within a few minutes converge with the original stalk into a single aperture. Interfering with apoptosis by overexpressing the P35 cell death inhibitor in imaginal and larval tissues does not affect epithelial perforation and disc eversion (Pastor-Pareja, 2004).

Following coalescence, the progressive widening of the hole by intercalation of PS cells at the leading front was observed. A cell lineage analysis was performed and multiple clones of PS cells were found, that remain compact up to the third instar larval stage in the PE and lose cohesion during eversion. Thus, PS cells appear to change neighbors, become extremely active, and emit and retract filopodia and lamellipodia at their front and rear ends. They squeeze in between themselves and the rest of the epithelium (planar intercalation), migrating to and expanding the front of the disc, and leading the migration over the larval tissue (Pastor-Pareja, 2004).

Simultaneous to wing disc eversion, all legs and haltere discs evert using the same mechanism (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).

The JNK signaling cascade dictates the expression of puc in all PS cells but their early specification appears not to be affected by lowering the level of JNK activity, since the complete absence of Hep function did not alter either their number or morphology in third instar larval discs. However, several mutant phenotypes have provided strong evidence for a leading role of the JNK pathway in imaginal disc fusion and disc eversion; e.g., hep mutants occasionally show uneverted wing discs lying inside the body of the pupa. When and how is JNK signaling needed? Transversal semi-thin sections, at 6 ± 1 hr APF, long after closure is completed in wild-type, of hep^r75 (a strong hypomorphic mutation) pupae show a range of phenotypic defects (classes I to III). Class I corresponds to a complete failure of PS/larval apposition (40% of individuals); in class II, discs apposed to the larval tissue but did not complete their eversion (50%); the mildest condition, class III, refers to discs that everted completely and advanced to some extent but were unable to fuse (10%). By the complete inactivation of the JNK signaling cascade through the ubiquitous overexpression of puc (from 48-60 hr before puparium formation onward), a fully penetrant failure was found of disc apposition to the larval epidermis (class I phenotype). A delayed or reduced (in a puc heterozygous background) overexpression of puc produced less severe class II and III phenotypes. Thus, the JNK cascade appears to be essential for PS and larval cell apposition and, as suggested by the observed phenotypic progression, may also be involved in later steps during eversion (Pastor-Pareja, 2004).

JNK activity levels also affect the degree of PEMT in PS cells. ZAs are absent from leading front cells and the membrane localization of DE-cadherin and Arm is progressively lost, as PS cells moved closer to the free edge. However, in hep^r75 mutant pupae (class III), the cells at the leading front of the disc do not delocalize either Arm or DE-cadherin in the free edge, suggesting that partial loss of JNK signaling blocks the correct transition of these cells from immotile epithelial to migrating and invading leading front cells. Further, a surplus of JNK activity in PS cells in puc^E69F-GAL4 mutants conveyed the transition of an excess of PS cells to a mesenchymatic phenotype. Hence, an adequate balance of JNK activity is key to control the level of PEMT. Too few mesenchymal-like PS cells restrain the ability of discs to evert and spread, while too many transformed cells affect the ability of discs to appropriately fuse. Further, puc^E69F-GAL4 mutants also showed enhanced cell motility and massive cell detachment from the free edges of the epithelium. These cells adopted a rounded shape but remained in close proximity, establishing transient contacts. Conversely, leading cells in hep^r75 mutants do not show any migratory activity. Hence, the JNK pathway regulates not only the adhesive properties of PS cells, but also their motility (Pastor-Pareja, 2004).

In summary, the JNK signaling cascade participates in four key steps in the process of disc eversion: (1) the expression of puc in PS cells; (2) the apposition of PS and larval cells; (3) the regulation of the adhesive and motile properties of PS cells as they undergo PEMT, and (4) the maintenance of the adhesion between the larval and imaginal tissue (Pastor-Pareja, 2004).

Thus, within the first 5 hr after puparium formation, the precursors of the adult structures evert. Multiple evidences show that eversion is mediated by actin microfilaments contraction, which modulate a general change of morphology of PS and larval cells driving the inside-out eversion of the disc. Several observations, however, suggest that other morphogenetic mechanisms are also involved (Pastor-Pareja, 2004).

(1) An imaginal disc is a rigidly determined primordium, which allows the construction of fate maps. Surprisingly, peripodial fate maps of Calliphora, a related diptera, show that adjacent territories develop into nonadjacent adult pleural structures, suggesting that the peripodial layer splits during metamorphosis (Pastor-Pareja, 2004).

(2) PS cells expressing puc relocate during eversion to the leading front. Thus, intercalation of PS cells appears to be concurrent to eversion (Pastor-Pareja, 2004).

(3) Pupal serial sectioning shows that, at eversion, imaginal discs appose to the larval epidermis through their peripodial side. Just before eversion, PS cells lose their basal lamina and detach from the extracellular matrix (Pastor-Pareja, 2004).

(4) Preceding disc eversion, in vivo time-lapse reveals the opening of larval/peripodial gaps, which are the outcome of the invasive behavior and planar intercalation (PEMT) of PS cells (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).

Puckered, a Drosophila MAPK phosphatase, ensures cell viability by antagonizing JNK-induced apoptosis

MAPK phosphatases (MKPs) are important negative regulators of MAPKs in vivo, but ascertaining the role of specific MKPs is hindered by functional redundancy in vertebrates. MKP function was characterized by examining the function of Puckered (Puc), the sole Drosophila Jun N-terminal kinase (JNK)-specific MKP, during embryonic and imaginal disc development. Puc is a key anti-apoptotic factor that prevents apoptosis in epithelial cells by restraining basal JNK signaling. Furthermore, JNK signaling plays an important role in gamma-irradiation-induced apoptosis, and this study examined how JNK signaling fits into the circuitry regulating this process. Radiation upregulates both JNK activity and puc expression in a p53-dependent manner; apoptosis induced by loss of Puc can be suppressed by p53 inactivation. JNK signaling acts upstream of both Reaper and effector caspases. JNK signaling directs normal developmentally regulated apoptotic events. However, if cell death is prevented, JNK activation can trigger tissue overgrowth. Thus, MKPs are key regulators of the delicate balance between proliferation, differentiation and apoptosis during development (McEwen, 2005).

Mammalian JNK signaling promotes apoptosis in response to both extrinsic (e.g. TNF) and intrinsic (e.g. DNA damage) cues. Drosophila JNK signaling plays a similar role in apoptosis triggered by extrinsic cues, but its role in response to intrinsic cues remains to be established. The role of Puc in regulating responses to either sort of signal has also been unclear (McEwen, 2005).

This study found that removal of Puc triggers apoptosis in epithelia, even in the absence of stress. Thus, basal JNK signaling exists in the absence of stress in embryonic and larval epithelia, and if Puc is not present to restrain this intrinsic JNK activity, it exceeds a threshold and triggers apoptosis. This suggests cells may regulate apoptosis without exogenous JNK stimulation, by regulating MKP levels. Biochemical studies suggest a possible mechanism. Several MKPs are labile proteins, whose half-lives can be shortened or lengthened by post-translational modification. Thus, cell signals may influence apoptosis by modulating MKP accumulation (McEwen, 2005).

Although Puc is crucial to restrain JNK activity and prevent apoptosis in most epithelia, high-level JNK signaling does not always induce apoptosis. JNK signaling is required for embryonic dorsal closure. High JNK activity is normally restricted to the dorsal-most epithelial cells, though in puc mutants it extends into adjacent cells. However, this JNK signaling does not trigger apoptosis. By contrast, ectopic JNK signaling in ventral and lateral epithelial cells in puc mutants does trigger apoptosis. Thus, cells where JNK activity is normally high are somehow refractory to JNK-induced apoptosis. JNK signaling normally activates Dpp in dorsal epithelial cells, with ectopic Dpp activation in more lateral cells in puc mutants -- perhaps this is anti-apoptotic, since Dpp is a survival factor in wing discs. Interplay between death and survival signals may also explain the strongly synergistic stimulation of apoptosis in the embryonic epidermis observed when zygotic Puc (lowering the threshold for JNK signaling) and the survival signal Wg are simultaneously eliminated (McEwen, 2005).

Studies of JNK signaling in mammalian cells suggest that it plays a role in radiation-induced apoptosis. This hypothesis was tested in Drosophila. When JNK activation was blocked by expressing Myc-Puc using a JNK-independent promoter, radiation-induced apoptosis is attenuated. Thus, JNK signaling plays a crucial role in promoting apoptosis in response to radiation in Drosophila, paralleling its essential role in mammalian UV-induced apoptosis (McEwen, 2005).

In mammalian cells, prolonged JNK activity correlates with apoptosis, while transient activation induces cell proliferation. Thus, differences in both signal amplitude and duration are key determinants of the biological outcome. One mechanism modulating signal amplitude is a negative feedback loop; JNK signaling induces expression of MKPs such as Puc. These data provide an instance of how this may regulate the DNA damage response. puc is upregulated by gamma-irradiation in a JNK-dependent manner, and artificially prolonged Myc-Puc expression prevents radiation-induced apoptosis. Thus, the initial increase in Puc expression following irradiation may create a 'grace period' during which Puc elevates the threshold of JNK activation required to induce apoptosis. During this time, cells could attempt to repair radiation-induced damage. However, if damage persists and JNK stimulation continues, the Puc-defined threshold may be exceeded. Indeed, artificially prolonged p53 overexpression overcomes the anti-apoptotic effects of Puc overexpression, perhaps overwhelming the ability of Puc to restrain JNK (McEwen, 2005).

p53 is a key regulator of decisions between life and death in response to DNA damage. Thus, how JNK signaling and p53 are integrated was investigated. Expression of p53 activates JNK, as revealed by its effect on a JNK enhancer trap reporter JNK^REP, while loss of p53 prevents radiation-induced JNK activation. These results suggest that p53 acts upstream of JNK signaling in response to cellular stress. Several mechanisms are possible; e.g., p53 might upregulate transcription of JNK pathway components, whose overexpression can induce apoptosis (McEwen, 2005).

p53 normally monitors genome integrity. Loss of p53 significantly, although not completely, suppresses apoptosis induced by Puc inactivation. One model to explain this suggests that basal levels of DNA damage or replication errors act through p53 to regulate basal JNK activity during normal development. This basal activity is kept below the apoptotic threshold by Puc. In p53 mutants, basal JNK activity would be lowered enough so that it could not exceed the apoptotic threshold, even in the absence of Puc. Alternatively, p53 may also function downstream of JNK. Consistent with this, p38 can activate p53 in response to UV, and JNK can regulate p53 stability/activity via direct phosphorylation. This would set up a positive-feedback loop: DNA damage-triggered activation of p53 would induce JNK activation, which would further elevate p53 activity. Additional experiments are required to test these alternate hypotheses (McEwen, 2005).

Signal transduction pathways regulate apoptosis, at least in part, by regulating transcription of rpr, hid and grim (the RHG proteins), the key developmental effectors of apoptosis in Drosophila. The relationship between JNK signaling, RHG proteins and caspase activation are not well understood. Caspase 3 cleavage of Mst1, an upstream regulator of JNK and p38, has been suggested to amplify apoptotic responses, while other data suggest that Mst1 activates caspases via a JNK-dependent pathway. Likewise, in Drosophila, initiation of an apoptotic response by inactivation of DIAP1 may lead to caspase-independent induction of a JNK^REP (McEwen, 2005).

Regulatory relationships between JNK activation, RHG proteins and caspase activation were directly assessed. The data suggest that JNK signaling acts through RHG proteins and caspases to induce apoptosis. JNK signaling is required for rpr-reporter induction in response to radiation. Thus, RHG proteins may be JNK-responsive target genes upregulated to elicit apoptosis. Consistent with this hypothesis, mis-expression of eiger, a known JNK activator, has been shown to promote hid expression and apoptosis in eye discs (McEwen, 2005).

To assess whether JNK signaling can be triggered by caspase activation, JNK^REP activity was examined in response to Rpr or Hid expression. Both induce apoptosis without concomitant JNK activation. Furthermore, these data suggest that, in at least some contexts, caspases act downstream of JNK: p35-mediated caspase blockade allows cells with elevated JNK signaling to survive in imaginal discs, but does not prevent JNK activation in response to irradiation. Thus, RHG proteins elicit caspase-mediated apoptosis downstream of JNK activation (McEwen, 2005).

Work on JNK-induced apoptosis in Drosophila has focused on its role in the complex processes shaping organ size. Wing disc cells make decisions about whether to die or proliferate by integrating levels of different developmental signals they receive, and comparing their status with that of their neighbors. This occurs in part by competition for survival signals like the TGFbeta family member Dpp. Complex crosstalk among this and other signaling pathways precisely regulates the size and pattern of the wing, in a process that is very resistant to tissue damage or developmental errors. Discontinuities in smooth morphogen gradients, which may arise from errors in patterning or tissue injury, are corrected in part by JNK-dependent apoptosis (McEwen, 2005).

These data clarify roles for JNK signaling in adult development. Failure to recover puc clones anywhere in the wing disc suggests that basal JNK activity is sufficient to promote cell-autonomous apoptosis independent of other signals activating JNK. However, JNK signaling does not play a crucial role in wing patterning; JNK inactivation in the posterior compartment (by Myc-Puc mis-expression) does not have drastic consequences. Roles for JNK signaling can be identified in genitalia and thoracic bristles, where it regulates developmentally programmed apoptosis (McEwen, 2005).

Can MKPs act as tumor suppressors? JNK signaling plays complex roles during oncogenesis. It can prevent tumorigenesis by promoting apoptosis, and it can promote tumorigenesis by supporting Ras-mediated or BCR-Abl-mediated transformation. Since each tumor type has a unique set of mutations in oncogenes and/or tumor suppressors, the phenotypic effects of JNK activation probably differ depending on the activity of other pathways (McEwen, 2005).

Inhibition of apoptosis is one prerequisite for tumorigenesis. Thus the consequences of JNK activation were examined when apoptosis was blocked. When cell death was blocked in the posterior compartment of the wing and the restraints on JNK activation were relaxed by puc heterozygosity, tissue overgrowth occurred. Groups of posterior cells, presumably of clonal origin, exhibited elevated levels of JNK activation, and formed small overgrowths both in developing imaginal discs and in the resulting adult wings and legs. Thus, when apoptosis is suppressed, JNK activation can lead to tissue over-growth (McEwen, 2005).

Related results have been reported with respect to inducing apoptosis by diap inactivation, irradiation or Hid expression, while simultaneously blocking caspase activation. This triggers non-autonomous proliferation of neighboring cells, presumably to compensate for cell lost by apoptosis. Furthermore, some of the 'undead' cells produced by these treatments show JNK-dependent upregulation of Wg or Dpp. Wg signaling has been shown to be required for compensatory proliferation. The current results extend previous studies, since the current experiment did not actively induce apoptosis, but simply blocked caspase activation in puc heterozygotes. Thus, when apoptosis is inhibited and Puc repression is reduced, cells become susceptible to runaway JNK activation. It is likely that analogous focal JNK activation occurs in cells in which apoptosis is not blocked (e.g., anterior compartment cells in these experiments), but JNK-induced apoptosis rapidly eliminates them. At least a subset of the undead cells activate expression of Wg, which may promote excess growth. Interestingly, however, this ectopic Wg expression does not alter cell fates, at least in those situations where overgrowths survive to be observed in the adult wing (McEwen, 2005).

Together, these data have interesting implications. In tumor cells in which apoptosis is prevented, JNK signaling might switch from promoting apoptosis to promoting proliferation by inducing Wnt or TGFbeta, suggesting how JNK signaling can be both pro- and anti-tumorigenic. Future experiments should address mechanisms by which JNK activation triggers Wnt and TGFβ signaling. These data also suggest that runaway JNK activation can promote cell-autonomous proliferation, since groups of cells with elevated JNK activity are associated with overgrowths. Thus, JNK activation or loss of MKP-mediated JNK repression may play multiple roles in tumors whose cells have lost the ability to die (McEwen, 2005).

Ectopic JNK activation occurred in small groups of cells in random positions throughout the posterior compartment. The event(s) that initiate unrestrained JNK activation in these cells remain to be defined. Perhaps there are stochastic variations in JNK signaling that are normally below the threshold triggering apoptosis. However, when Puc activity is reduced, some cells may exceed this threshold, triggering runaway JNK activation in the initial cell and its descendents. Variations in JNK activity may also be induced by spontaneous DNA damage. Normally, such damage would trigger JNK-dependent apoptosis; however, if apoptosis is blocked, the cells proliferate. Finally, loss-of-heterozygosity at the puc locus could create cells lacking restraints on JNK signaling. Since mitotic recombination in somatic tissue can occur, Puc might act in a manner analogous to classic tumor suppressor genes. Additional studies will be required to distinguish between these possibilities (McEwen, 2005).

In summary, this work establishes that Puc is a key negative regulator of apoptosis throughout Drosophila development. In its absence, basal JNK activity is poised to eliminate cells from the developing epithelium. The results position JNK signaling in the hierarchy of events regulating radiation-induced apoptosis. Finally, the data support the possibility previously suggested by cytogenetics that MKPs may act as tumor suppressor genes. These data prompt many new mechanistic questions regarding the role of JNK signaling in apoptosis and oncogenesis (McEwen, 2005).

Loss of cell polarity drives tumor growth and invasion through JNK activation in Drosophila

Apparent defects in cell polarity are often seen in human cancer. However, the underlying mechanisms of how cell polarity disruption contributes to tumor progression are unknown. Using a Drosophila genetic model for Ras-induced tumor progression, a molecular link has been shown between loss of cell polarity and tumor malignancy. Mutation of different apicobasal polarity genes activates c-Jun N-terminal kinase (JNK) signaling and downregulates the E-cadherin/β-catenin adhesion complex, both of which are necessary and sufficient to cause oncogenic Ras^V12-induced benign tumors in the developing eye to exhibit metastatic behavior. Furthermore, activated JNK and Ras signaling cooperate in promoting tumor growth cell autonomously, since JNK signaling switches its proapoptotic role to a progrowth effect in the presence of oncogenic Ras. The finding that such context-dependent alterations promote both tumor growth and metastatic behavior suggests that metastasis-promoting mutations may be selected for based primarily on their growth-promoting capabilities. Similar oncogenic cooperation mediated through these evolutionarily conserved signaling pathways could contribute to human cancer progression (Igaki, 2006).

Most human cancers originate from epithelial tissues. These epithelial tumors, except for those derived from squamous epithelial cells, normally exhibit pronounced apicobasal polarity. However, these tumors commonly show defects in cell polarity as they progress toward malignancy. Although the integrity of cell polarity is essential for normal development, how cell polarity disruption contributes to the signaling mechanisms essential for tumor progression and metastasis is unknown. To address this, a recently established Drosophila model of Ras-induced tumor progression triggered by loss of cell polarity has been used. This fly tumor model exhibits many aspects of metastatic behaviors observed in human malignant cancers, such as basement membrane degradation, loss of E-cadherin expression, migration, invasion, and metastatic spread to other organ sites (Pagliarini, 2003). In the developing eye tissues of these animals, loss of apicobasal polarity is induced by disruption of evolutionarily conserved cell polarity genes such as scribble (scrib), lethal giant larvae (lgl), or discs large (dlg), three polarity genes that function together in a common genetic pathway, as well as other cell polarity genes such as bazooka, stardust, or cdc42. Oncogenic Ras (Ras^V12), a common alteration in human cancers, causes noninvasive benign overgrowths in these eye tissues (Pagliarini, 2003). Loss of any one of the cell polarity genes somehow strongly cooperates with the effect of Ras^V12 to promote excess tumor growth and metastatic behavior. However, on their own, clones of scrib mutant cells are eliminated during development in a JNK-dependent manner; expression of Ras^V12 in these mutant cells prevents this cell death (Igaki, 2006).

To better quantify the metastatic behavior of tumors in different mutant animals, the analysis focused on invasion of the ventral nerve cord (VNC), a process in which tumor cells leave the eye-antennal discs and optic lobes (the areas where they were born) and migrate to and invade a different organ, the VNC. It was further confirmed that the genotypes associated with the invasion of the VNC in this study also resulted in the presence of secondary tumor foci at distant locations, although the number and size of these foci were highly variable (Pagliarini, 2003; Igaki, 2006).

In analyzing the global expression profiles of noninvasive and invasive tumors induced in Drosophila developing eye discs, it was observed that expression of the JNK phosphatase puckered (puc) was strongly upregulated in the invasive tumors. Upregulation of puc represents activation of the JNK pathway in Drosophila. Therefore an enhancer-trap allele, puc-LacZ, was used to monitor the activation of JNK signaling in invasive tumor cells. Strong ectopic JNK activation was present in invasive tumors, while only a slight expression of puc was seen in restricted regions of Ras^V12-induced noninvasive overgrowth. Intriguingly, more intense JNK activation was seen in tumor cells located in the marginal region of the eye-antennal disc and tumor cells invading the VNC. Analysis of clones of cells with a cell polarity mutation alone revealed that JNK signaling was activated by mutation of cell polarity genes. Notably, JNK signaling was not activated in a strictly cell-autonomous fashion. JNK activation in these cells was further confirmed by anti-phospho-JNK antibody staining that detects activated JNK (Igaki, 2006).

To examine the contribution of JNK activation to metastatic behavior, the JNK pathway was blocked by overexpressing a dominant-negative form of Drosophila JNK (Bsk^DN). As previously reported (Pagliarini, 2003), clones of cells mutant for scrib, lgl, or dlg do not proliferate as well as wild-type clones, while combination of these mutations with Ras^V12 expression resulted in massive and metastatic tumors. Strikingly, inhibition of JNK activation by Bsk^DN completely blocked the invasion of the VNC, as well as secondary tumor foci formation. Drosophila has two homologs of TRAF proteins (DTRAF1 and DTRAF2), which mediate signals from cell surface receptors to the JNK kinase cascade in mammalian systems. It was found that RNAi-mediated inactivation of DTRAF2, but not DTRAF1, in the tumors strongly suppressed their metastatic behavior. Inactivation of dTAK1, a Drosophila JNK kinase kinase (JNKKK), or Hep, a JNKK, also suppressed metastatic behavior. Drosophila has two known cell surface receptors that act as triggers for the JNK pathway, Wengen (TNF receptor) and PVR (PDGF/VEGF receptor). Intriguingly, it was found that RNAi-mediated inactivation of Wengen partially suppressed tumor invasion. Inactivation of PVR, in contrast, did not show any suppressive effect on metastatic behavior. It was also found that the metastatic behavior of Ras^V12-expressing tumors that were also mutated for one of three other cell polarity genes, bazooka, stardust, or cdc42, was also blocked by Bsk^DN. These data indicate that loss of cell polarity contributes to metastatic behavior by activating the evolutionarily conserved JNK pathway (Igaki, 2006).

Next, whether JNK activation is sufficient to trigger metastatic behavior in Ras^V12-induced benign tumors was examined. Two genetic alterations can be used to activate JNK in Drosophila. First, JNK signaling can be activated by overexpression of Eiger, a Drosophila TNF ligand. While mammalian TNF superfamily proteins activate both the JNK and NFκB pathways, Eiger has been shown to specifically activate the JNK pathway through dTAK1 and Hep. Indeed, the eye phenotype caused by Eiger overexpression could be reversed by blocking JNK through Bsk-IR (Bsk-RNAi). Second, overexpression of a constitutively activated form of Hep (Hep^CA) can also activate JNK signaling. However, the eye phenotype caused by Hep^CA overexpression was only slightly suppressed by Bsk-IR, suggesting that Hep^CA overexpression may have additional effects other than JNK activation. Therefore Eiger overexpression was used to activate JNK in Ras^V12-induced benign tumors, and it was found that the Ras^V12+Eiger-expressing tumor cells did not result in the invasion of the VNC. This indicates that loss of cell polarity must induce an additional downstream effect(s) essential for metastatic behavior. A strong candidate for the missing event is downregulation of the E-cadherin/catenin adhesion complex, since this complex is frequently downregulated in malignant human cancer cells and is also downregulated by loss of cell polarity genes in Drosophila invasive tumors (Pagliarini, 2003). In addition, it has been recently reported that higher motility of mammalian scrib knockdown cells can be partially rescued by overexpression of E-cadherin-catenin fusion protein, suggesting a role of E-cadherin in preventing polarity-dependent invasion. Furthermore, overexpression of E-cadherin blocks metastatic behavior of Ras^V12/scrib^−/− tumors (Pagliarini, 2003), indicating that loss of E-cadherin is essential for inducing tumor invasion in this model. It was found that loss of the Drosophila E-cadherin homolog shotgun (shg), combined with the expression of both Ras^V12 and Eiger, induced the invasion of the VNC. Intriguingly, loss of shg in Ras^V12-expressing clones also showed a weak invasive phenotype at lower penetrance. In agreement with the essential role of JNK in tumor invasion, clones of shg^−/− cells weakly upregulated puc expression. It was further found that JNK activation in dlg^−/− clones is not blocked by overexpression of E-cadherin, suggesting that mechanism(s) other than loss of E-cadherin also exist for inducing JNK activation downstream of cell polarity disruption. The metastatic behavior of Ras^V12+Eiger/shg^−/− tumors was completely blocked by coexpression of Bsk^DN, indicating a cell-autonomous requirement of JNK activation for this process. Furthermore, it was found that loss of the β-catenin homolog armadillo also induced metastatic behavior in Ras^V12-induced benign tumors. In contrast, overexpression of Hep^CA in Ras^V12/shg^−/− cells resulted in neither enhanced tumor growth nor metastatic behavior. Together, these results suggest that, although the Ras^V12+Eiger/shg^−/− does not completely phenocopy the effect of Ras^V12/scrib^−/−, activation of JNK signaling and inactivation of the E-cadherin/catenin complex are the downstream components of cell polarity disruption that trigger metastatic behavior in Ras^V12-induced benign tumors (Igaki, 2006).

Multiple protein phosphatases are required for mitosis in Drosophila

Approximately one-third of the Drosophila kinome has been ascribed some cell-cycle function. However, little is known about which of its 117 protein phosphatases (PPs) or subunits have counteracting roles. This study investigated mitotic roles of PPs through systematic RNAi. It was found that G2-M progression requires Puckered, the JNK MAP-kinase inhibitory phosphatase and PP2C in addition to string (Cdc25). Strong mitotic arrest and chromosome congression failure occurs after Pp1-87B downregulation. Chromosome alignment and segregation defects also occurs after knockdown of PP1-Flapwing, not previously thought to have a mitotic role. Reduction of several nonreceptor tyrosine phosphatases produced spindle and chromosome behavior defects, and for corkscrew, premature chromatid separation. RNAi of the dual-specificity phosphatase, Myotubularin, or the related Sbf 'antiphosphatase' resulted in aberrant mitotic chromosome behavior. Finally, for PP2A, knockdown of the catalytic or A subunits led to bipolar monoastral spindles, knockdown of the Twins B subunit led to bridged and lagging chromosomes, and knockdown of the B' Widerborst subunit led to scattering of all mitotic chromosomes. Widerborst is associated with MEI-S332 (Shugoshin) and is required for its kinetochore localization. This study has identified cell-cycle roles for 22 of 117 Drosophila PPs. Involvement of several PPs in G2 suggests multiple points for its regulation. Major mitotic roles are played by PP1 with tyrosine PPs and Myotubularin-related PPs having significant roles in regulating chromosome behavior. Finally, depending upon its regulatory subunits, PP2A regulates spindle bipolarity, kinetochore function, and progression into anaphase. Discovery of several novel cell-cycle PPs identifies a need for further studies of protein dephosphorylation (Chen, 2007).

P2A is a heterotrimeric serine/threonine phosphatase composed of invariant catalytic (C) and structural (A) subunits together with one member of a family of B regulatory subunits thought to direct the AC core to different substrates. The Drosophila gene for the catalytic subunit of type 2A protein serine/threonine phosphatase (PP2A) is known as microtubule star (mts) because mutant embryos show multiple individual centrosomes with disorganized astral arrays of microtubules. In agreement with this mutant phenotype, it was found that S2 cells depleted for Mts (PP2A-C) displayed aberrant elongated arrays of microtubules with a high proportion (5- to 10-fold increase over the control) of bipolar monoastral spindles or monopolar spindles emanating from a single centrosomal mass. This phenotype is also consistent with the observations in Xenopus egg extracts where mitotic microtubules grow longer and bipolar spindles can not be assembled after inhibition of PP2A by low concentrations of okadaic acid (OA). It is speculated that the monopolar spindle phenotype after mts dsRNA treatment is a consequence of the spindle collapse rather than a failure in centrosome duplication or separation because most of the RNAi-treated cells showed well-separated centrosomes during prophase. In support of this view, spindle bipolarity can be rescued by restoration of microtubule dynamics in OA-treated Xenopus egg extracts (Chen, 2007).

In Drosophila, as in many other eukaryotes, mitosis-specific phosphorylation of histone H3 requires Aurora B activity, but the identity of the opposing phosphatase remains unclear. Because P-H3 (Ser 10) levels were used for monitoring the mitotic index in this analysis, it is possible that a high mitotic index observed after RNAi for PPs may also reflect a defect in dephosphorylating P-H3 in the absence of PPs upon mitotic exit. The phosphorylation state of this histone was therefore studied after RNAi for PPs that displayed a significant increase in the mitotic index in the screen. The immunostaining of control cells showed that P-H3 signals began to decrease at early telophase and then disappeared completely at late telophase. After RNAi knockdown of Mts (PP2A-C) or Pp1-87B, however, the majority of mitotic cells were arrested at prometaphase, but late telophase figures could occassionally be found showing an abnormal accumulation of P-H3 on decondensed chromosomes. To better assess the effect of depletion of these two PPs on P-H3 dephosphorylation, the spindle-assembly checkpoint was inactivated by simultaneously knocking down BubR1. It was found that this rescued the prometaphase arrest of cells simultaneously depleted for Mts or Pp1-87B; this allowed a study of telophase cells. P-H3 was present in the majority of such telophase cells compared to control cells, indicating that both PPs are required for P-H3 dephosphorylation. These results are in accordance with previous studies showing that reduction of PP1 activity can partially suppress defects in the mitotic histone H3 phosphorylation in yeast and C. elegans (Chen, 2007).

Downregulation of Pp2A-29B, the structural A subunit, revealed almost identical aberrant phenotypes to those observed after mts (PP2A-C) RNAi. Consistently, knockdown of Pp2A-29B (PP2A-A) led to a reduction of the protein level of Mts (PP2A-C) (Chen, 2007).

The Drosophila genome contains 4 B-type PP2A regulatory subunits, twins/tws/aar (B sub-type), widerborst/wdb (B' sub-type), Pp2A-B' (B' sub-type), and Pp2A-B" (B" sub-type), but mitotic defects have so far only been reported for mutants of tws. Consistent with the phenotype of tws mutants, it was observed that RNAi for this gene led to an increased proportion of anaphase figures showing lagging chromosomes and chromosome bridges (Chen, 2007).

In metazoans, the B' regulatory subunits of PP2A have evolved into two related subclasses with conserved central regions and diverged amino and carboxy termini. The protein encoded by widerborst (wdb) is more closely related to the human α and ɛ subunits (79%–80% identity) than to the β, γ, or δ subunits (69%–75% identity). Whereas RNAi for tws led to lagging chromosomes, wdb RNAi led to dramatic scattering of chromosomes throughout the spindle. Whether this dramatic effect of wdb RNAi on chromosome segregation reflected any particular subcellular localization of this regulatory subunit was examined. To this end, a GFP-tagged Wdb was expressed in S2 cells. During interphase and prophase, Wdb::GFP partially colocalized with the centromeric marker CID (CENP-A). After spindle formation, Wdb::GFP was found adjacent and external to the centromeres. Although less pronounced, this distribution remained during chromosome segregation at anaphase. Because MEI-S332 (Drosophila Shugoshin) is a dynamic centromeric marker, its distribution was examined in wdb RNAi cells. In control cells, MEI-S332 localized in a band between each pair of the centromeres at metaphase. After downregulation of wdb, however, greatly reduced MEI-S332 staining was found on the metaphase chromosome. In contrast, depletion of MEI-S332 by RNAi did not affect the normal localization of the Wdb B' PP2A subunit. Thus, it is concluded that the Wdb B' subunit is required for correct localization of MEI-S332 but not vice versa. Whether the two proteins existed in the same complex was examined. To address this, a Protein A (PrA)-tagged form of MEI-S332 was expressed in S2 cells to purify potential protein complexes and identify its components by mass spectrometry. The catalytic C (Mts), the structural A (PP2A-29B), and the regulatory B' (Wdb) and B (Tws) subunits of PP2A were identified associated with MEI-S332. Three recent studies also identified PP2A complexed to the B' subunit bound to Shugoshin (Sgo) in human and yeast cells, where they are thought to protect centromeric cohesin subunits from phosphorylation that would promote premature sister-chromatid separation. As with the archetypal family member, Drosophila MEI-S332, the Shugoshins function primarily to protect sister chromatids from separation in the first meiotic division but are also present in mitotic divisions. Consistent with these observations in Drosophila S2 cells, it has been found that depletion of PP2A in human cells led to premature dissociation of Shugoshin 1 (Sgo1) from the kinetochore and loss of mitotic centromere cohesion. The finding of Shugoshin complexed to PP2A/B' in yeast and human, and now in Drosophila, points to a highly evolutionally conserved role for this particular PP2A heterotrimer in regulating sister-chromatid cohesion. Interestingly, Tws B regulatory subunit was also recovered associated with MEI-S332. How this subunit of PP2A might function with MEI-S332 should be the subject of future investigations (Chen, 2007).

Only a moderatedly elevated mitotic index (by approximately 10%) was observed after downregulation of the second Drosophila B' regulatory subunit (Pp2A-B'/B56-1). However, when this second B' subunit was simultaneously knocked down with Wdb, this led to similar phenotypes seen in Mts (PP2A-C) or Pp2A-29B (PP2A-A)-depleted cells. Western-blot analysis showed that the Mts (PP2A-C) level decreased after simultaneous knockdown of both B' subunits, suggesting that this phenotype could be partially due to the loss of PP2A catalytic subunit, although the possibility that the two B' subunits share partially redundant mitotic functions cannot be excluded (Chen, 2007).

Cell-cycle kinases represent a large family of enzymes governing the cell division cycle. It is therefore not surprising that a considerable number of counteracting cell-cycle phosphatases (19% of the genes for tested) were identified in the current study. In addition to finding all the well-known PPs required for cell-cycle progression in Drosophila (Mts, Tws, String, Pp4-19C, and Pp1-87B), the Drosophila counterparts of some eight PPs implicated in cell-cycle functions were identified from studies on other organisms together with six PPs for which novel cell-cycle roles were ascribe. These results were validated by confirming the observed phenotypes with a second nonoverlapping dsRNA. In two cases (flw and csw), their mitotic roles were confirmed through the analysis of phenotypes in mutant larval neuroblasts. The RNAi phenotypes of catalytic subunits were evaluated by observing similar phenotypes after downregulation of the corresponding regulatory subunits (e.g., Pp4-19C and PPP4R2r, Mts/PP2A-C and Pp2A-29B/PP2A-A, and simultaneous RNAi of the two PP2A-B' regulatory subunits). Although a recent large-scale RNAi screen based solely on flow cytometry in Drosophila S2 cells identified many regulators of the cell cycle, cell size, and cell death, this study sho