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).

Cell death-induced regeneration in wing imaginal discs requires JNK signalling

Regeneration and tissue repair allow damaged or lost body parts to be replaced. After injury or fragmentation of Drosophila imaginal discs, regeneration leads to the development of normal adult structures. This process is likely to involve a combination of cell rearrangement and compensatory proliferation. However, the detailed mechanisms underlying these processes are poorly understood. A system was established to allow temporally restricted induction of cell death in situ. Using Gal4/Gal80 and UAS-rpr constructs, targeted ablation of a region of the disc could be performed and regeneration monitored without the requirement for microsurgical manipulation. Using a ptc-Gal4 construct to drive rpr expression in the wing disc resulted in a stripe of dead cells in the anterior compartment flanking the anteroposterior boundary, whereas a sal-Gal4 driver generated a dead domain that includes both anterior and posterior cells. Under these conditions, regenerated tissues were derived from the damaged compartment, suggesting that compartment restrictions are preserved during regeneration. These studies reveal that during regeneration the live cells bordering the domain in which cell death was induced first display cytoskeletal reorganisation and apical-to-basal closure of the epithelium. Then, proliferation begins locally in the vicinity of the wound and later more extensively in the affected compartment. Finally, regeneration of genetically ablated tissue was shown to require JNK activity. During cell death-induced regeneration, the JNK pathway is activated at the leading edges of healing tissue and not in the apoptotic cells, and is required for the regulation of healing and regenerative growth (Bergantiños, 2010).

Two main conclusions can be drawn from this work: (1) that genetically induced regeneration entails compartment-specific proliferation; and (2), that this type of regeneration requires JNK signalling for early regeneration events (Bergantiños, 2010).

This study established that the proliferation response to ptc>rpr induction is concentrated in the A compartment and consists of two activities: a local and a compartment-associated response. The local proliferation response resembles the activity of blastemas, a feature found in discs after fragmentation and implantation. The late compartment-restricted proliferation could be indicative of a reutilisation of developmental programs. The entire A compartment responds to the lack of the original ptc region by reactivating proliferation in order to achieve the final organ size. Thus, it is concluded that genetically induced regenerating discs restore the overall organ size by activation of proliferation, not only near the wound, as in fragmented and implanted discs, but also in the whole affected compartment. Thus, it is believed that the local proliferation is a fast and early response to the lost structures and that the later compartment-associated proliferation is a response to adjust the size of the tissue (Bergantiños, 2010).

ptc and sal were selected because of the precise removal of cells and also because they enabled testing whether both A and P compartments are involved in regeneration. The results suggest that when the A compartment is damaged (ptc>rpr), the P compartment only responds to the injury by sealing the gap that separates it from the A compartment through the generation of F-actin-rich cell extensions. These are projected to anchor the extensions from the cells at the edge of the A compartment as they proceed towards recovery of the intact cell sheet. In this situation, the regenerated tissue is derived exclusively from the A compartment. By contrast, when cells from both the A and P compartments are killed (sal>rpr), proliferation increases in both compartments. The boundaries between compartments are rapidly re-established after injury and prevent cells from crossing into adjacent compartments. Thus, boundaries are respected and compartments act as units of growth during regeneration (Bergantiños, 2010).

Following genetic ablation driven by either the ptc or sal drivers, healing starts at the DV boundary and spreads laterally towards the proximal regions, which are the last to close the wound. Cells at the DV boundary are arrested in G1-S, through a mechanism based on Notch and wg signalling. These arrested cells are the first to respond to healing and drive the cytoskeletal machinery for tissue reorganisation. This is consistent with the idea that the requirements for cell proliferation and for cell shape changes that occur during normal fly and vertebrate development and wound repair place incompatible demands on the cytoskeletal machinery of the cell. Another issue to be considered is that the DV boundary is the first zone of closure for F-actin extensions. This is reminiscent of Drosophila embryonic dorsal closure and wound repair, in which matching filopodia on both sides of the opening are recognised by the code of segment polarity genes in each parasegment. In addition, mechanical forces may be involved in tissue reorganisation. Stretching forces could be altered upon the induction of cell death, and they could have an important role in mounting a quick healing response. For example, mechanical forces, which have been proposed to act in the developing wing disc and compress the tissue through the central region, could stretch it towards the DV border after ablation of the ptc domain. Thus, either by matching affinities or by stretching forces, wound repair spreads from the apical DV border to basal and proximal domains (Bergantiños, 2010).

It has been shown in the Drosophila wing disc that massive loss of cells after irradiation gives rise to apparently normal adult wings as a result of compensatory proliferation driven by surviving cells. Experiments involving irradiation or induction of apoptosis in a p35 background have suggested that this compensatory proliferation is controlled by signals, including JNK, emerging from cells that have entered apoptosis, and that cell-death regulators, such as p53 and the caspase Dronc (Nedd2-like caspase), function as regulators of compensatory proliferation and blastema formation in the surviving cells. By contrast, the results show that proliferation is compartment specific and occurs independently of the dead tissue following targeted ablation. Two observations strongly support this interpretation. First, puc expression, as a marker of JNK activity, is concentrated in a narrow strip of apical cells, suggesting that JNK signalling is activated in the leading edges during wound closure. This again resembles other repair mechanisms described not only in imaginal discs, but also in other healing tissues, and reiterates epithelial fusion events observed in embryogenesis. Second, perturbation of the JNK pathway within the dying domain has no effect on either healing or regeneration. Even the early peak of localised mitosis near the wound and the later A-compartment-associated mitoses are present when UAS-bsk^DN and UAS-puc are driven in the dying domain. Effects on healing and regeneration are found only in hep mutant backgrounds, when JNK is impaired in the whole epithelium and not only in the dead domain. This requirement for the JNK pathway at the edges of the wound has also been found in studies of microsurgically induced regeneration. Cell lineage analysis of puc-expressing cells near the wound has shown that puc sets the limits of a blastema and that puc derivatives are able to reconstitute most of the missing tissue (Bergantiños, 2010).

Finally, whether JNK is required for healing alone or also functions as a signal for proliferation remains an open issue. Rapid local proliferation is affected in unhealed hep heterozygotes. Also, sal^PE>rpr wing regeneration cannot be achieved after 10 hours induction in a hep^r75 background. Reduced proliferation could be due to a lack of healing or to loss of JNK activity. The possibility canot be ruled out that the JNK cascade, through the active AP-1 (Kayak and Jun-related antigen -- FlyBase) transcription factor complex, targets not only genes required for healing and epithelial fusion, but also those required for regenerative growth. In mammals, inhibition of the JNK pathway or lack of c-Jun results in eyelid-closure defects and also impairs proliferation by targeting Egfr transcription. Reconstruction of normal pattern and size might also require multiple signals. It has recently been found that regenerative growth induced by cell death requires Wnt/Wg signalling to increase dMyc stability, suggesting the involvement of other signalling pathways and also cell competition. It is very likely that an integrated network of signals and cell behaviours is necessary to reconstitute the damaged tissue (Bergantiños, 2010).

Taken together, these results suggest a model for cell-induced regeneration that includes two phases. The first, which occurs near the wound edges, involves JNK activity and is important for healing and rapid local proliferation. The second involves proliferation to compensate for the lost tissue and is extended throughout the damaged compartment. As in normal development, the regenerative growth that occurs in this second phase requires the reconstitution of morphogenetic signals that drive proliferation (Bergantiños, 2010).

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 showed a very low degree of overlap with the cureent analysis (only six), reflecting the need for more sensitive small-scale screens that can examine the functional requirements of assayed proteins in greater detail. These results have provided novel insights into the cell-cycle functions of the Drosophila PPs, and it is likely that, in many cases, these functions have been conserved in other metazoans including humans. This study should guide future work aimed at elucidating the significance and mechanisms of the balanced activities of PKs and PPs in regulating the cell division cycle. The challenge ahead will be to match up the functions of the PPs that were identified with their corresponding counteracting PKs and to identify their common key substrates (Chen, 2007).

The nonmuscle myosin phosphatase PP1β (flapwing) negatively regulates Jun N-terminal kinase in wing imaginal discs of Drosophila

Drosophila flapwing (flw) codes for serine/threonine protein phosphatase type 1ß (PP1ß). Regulation of nonmuscle myosin activity is the single essential flw function that is nonredundant with the three closely related PP1α genes. Flw is thought to dephosphorylate the nonmuscle myosin regulatory light chain, Spaghetti Squash (Sqh); this inactivates the nonmuscle myosin heavy chain, Zipper (Zip). Thus, strong flw mutants lead to hyperphosphorylation of Sqh and hyperactivation of nonmuscle myosin activity. This study shows genetically that a Jun N-terminal kinase (JNK) mutant suppresses the semilethality of a strong flw allele. Alleles of the JNK phosphatase puckered (puc) genetically enhance the weak allele flw¹, leading to severe wing defects. Introducing a mutant of the nonmuscle myosin-binding subunit (Mbs) further enhances this genetic interaction to lethality. puc expression is upregulated in wing imaginal discs mutant for flw¹ and puc^A251, and this upregulation is modified by JNK and Zip. The level of phosphorylated (active) JNK is elevated in flw¹ enhanced by puc. Together, this study shows that disruption of nonmuscle myosin activates JNK and puc expression in wing imaginal discs (Kirchner, 2007; full text of article).

This study shows that the nonmuscle myosin phosphatase flw interacts genetically with components of the JNK signaling pathway. The proteins JNK and PP1ß (Flw), as well as the mechanisms of JNK signal transduction and nonmuscle myosin activation, are highly conserved between Drosophila and humans. This suggests that findings of Drosophila PP1ß regulating JNK through nonmuscle myosin may be relevant for similar processes in human cells and tissues (Kirchner, 2007).

bsk (JNK) mutants suppressed the semilethality of the strong allele flw⁶, while puc and constitutively active hep^CA enhanced the weak viable allele flw¹, resulting in severe wing defects. The level of diphosphorylated (active) JNK was elevated in a flw¹/Y ; puc^A251 mutant background. This, together with the finding that puc expression was upregulated in wing imaginal discs of flw¹/Y ; puc^A251/+, implies that flw can act as a negative regulator of JNK. This was further supported by the fact that puc expression was not upregulated in flw¹/Y ; bsk¹/+ ; puc^A251/+ wing imaginal discs, where reduced amounts of overall JNK (Bsk) protein probably compensate for elevated activity of JNK in flw¹/Y ; puc^A251/+. A possible difficulty with using puc^A251 (or puc^E69, another frequently used puc enhancer trap) as a reporter of puc expression is that both lines are also puc mutants, and puc probably regulates its own expression through a negative feedback loop involving JNK. However, it was confirmed in an independent assay with an anti-P-JNK antibody that JNK was indeed ectopically activated in flw¹/Y ; puc^A251/+. Wing imaginal disc extracts from both flw¹ and puc^A251/+ showed somewhat elevated levels of monophosphorylated, but not diphosphorylated, JNK. In flw¹/Y ; puc^A251/+, it is suggested that dephosphorylation of JNK fails to such an extent that JNK is substantially diphosphorylated and thereby activated. Since it has been shown that activation of JNK in wing imaginal discs induces apoptosis, it is likely that the flw¹/Y ; puc/+ wing phenotype is due to increased death of cells with aberrantly activated JNK (Kirchner, 2007).

A zip mutant suppresses the upregulation of puc in wing imaginal discs as well as the adult wing phenotype of flw¹/Y ; puc^A251/+. This shows that nonmuscle myosin acts upstream of JNK and mediates an activating signal on JNK in a flw¹/Y ; puc/+ mutant background; this is also consistent with the finding that a mutant in the myosin phosphatase-targeting subunit Mbs enhances flw¹/Y ; puc^A251/+ to lethality. Drosophila encodes two myosin phosphatase-targeting subunits, Mbs and MYPT-75D. Mbs binds both Flw and Pp1-87B, while MYPT-75D binds Flw specifically Vereshchagina, 2004). Unfortunately, it was not possible to test for genetic interaction between flw¹/Y ; puc^A251/+ and MYPT-75D because no MYPT-75D mutants have been described. It was also found that a Rho1 mutant abolishes ectopic lacZ staining in flw¹/Y ; puc^A251 wing imaginal discs. Rho1 is an activator of Rho-dependent kinase, which phosphorylates and activates myosin light chain, as well as phosphorylating and inhibiting Mbs. Furthermore, both zip and Rho1, in combination with other genetic interactors, have been reported to show a malformed third-leg phenotype that resembles that of flw¹/Y ; puc/+ (Kirchner, 2007).

Dorsal closure and wound healing depend on both nonmuscle myosin and JNK activity, but a clear genetic or molecular interaction between these pathways has not been previously demonstrated. This is the first study that shows that disruption of nonmuscle myosin can induce activation of JNK, although the mechanism of signal transduction is not clear. The single essential and nonredundant function of flw is the inhibition of nonmuscle myosin activity, presumably by dephosphorylating Sqh at T21 and S22. However, the results suggest that hyperphosphorylation of Sqh at these residues may not explain the elevated expression of puc in flw¹/Y ; puc^A251/+ flies. Additional mechanisms may exist to regulate nonmuscle myosin assembly and activity through phosphorylation, and the complex Flw/Mbs may have other targets in the actomyosin network in addition to Sqh. For example, moesin and focal adhesion kinase are potential targets for mammalian myosin phosphatase. Interestingly, overexpression of the focal adhesion protein tensin (blistery) in Drosophila wing imaginal discs activates JNK and induces apoptosis (Kirchner, 2007).

Two important questions remain unanswered regarding the results on the activation of JNK through nonmuscle myosin. (1) What is the molecular pathway from nonmuscle myosin to JNK? Several mechanisms have been identified that activate JNK and induce apoptosis in wing imaginal discs. For example, mutations in the caspase inhibitor DTraf1 (Drosophila tumor necrosis factor receptor-associated factor 1), as well as inhibition of DTraf1 by overexpression of Hid (head involution defective), Rpr (reaper), or Grim, induces JNK-mediated apoptosis, possibly through Msn (misshapen) or Ask1 (apoptosis signal-regulating kinase 1). Other factors involved in inducing apoptosis and activating JNK are Eiger (Drosophila tumor-necrosis factor superfamily ligand), the serine C-palmitoyltransferase Lace, Blistery (Drosophila Tensin), and Decapentaplegic and Wingless. It is not clear how these factors signal to JNK or whether they act in a single pathway, let alone whether any of them interact with nonmuscle myosin. It is likely that there are several independent ways of activating JNK, and it has been suggested that induction of apoptosis through JNK activation is a regulatory mechanism to eliminate abnormally developing cells in wing imaginal discs. (2)This leads directly to the second question: are the results of nonmuscle myosin signaling to JNK significant in a developmental context? The fact that bsk¹ suppresses the semilethality of flw⁶ indicates that the interactions that uncovered are not confined to the main experimental model of ectopic JNK activation in wing imaginal discs. The obvious system for studying possible interactions between nonmuscle myosin and JNK would be dorsal closure, which depends on both the coordinated assembly and the contraction of the actomyosin cytoskeleton and on activation of JNK. So far, there is no evidence that dorsal closure is affected in flw mutant embryos; however, there is a maternal contribution of flw that may conceal embryonic phenotypes. Actomyosin and JNK do not promote dorsal closure completely independently from each other; for example, the expression of many components of the actomyosin cytoskeleton is upregulated in response to JNK. Also, actin and myosin failed to accumulate along the leading edge of the epidermis in the puc mutant background. Both findings would place the actomyosin cytoskeleton downstream of JNK, whereas genetic data place flw and zip upstream of JNK and puc. It is possible, however, that actomyosin acts both upstream and downstream of JNK during dorsal closure. Because of the conserved nature of the components involved, it is likely that the finding that nonmuscle myosin can signal to and activate JNK is relevant to furthering the understanding of processes like dorsal closure and wound healing in Drosophila and humans (Kirchner, 2007).

D-JNK signaling in visceral muscle cells controls the laterality of the Drosophila gut

Although bilateral animals appear to have left–right (LR) symmetry from the outside, their internal organs often show directional and stereotypical LR asymmetry. The mechanisms by which the LR axis is established in vertebrates have been extensively studied. However, how each organ develops its LR asymmetric morphology with respect to the LR axis is still unclear. This study showed that Drosophila Jun N-terminal kinase (JNK) signaling is involved in the LR asymmetric looping of the anterior-midgut (AMG) in Drosophila. Mutant embryos of puckered (puc), which encodes a JNK phosphatase, show random laterality of the AMG. Directional LR looping of the AMG requires JNK signaling to be down-regulated by puc in the trunk visceral mesoderm. Not only the down-regulation, but also the activation of JNK signaling is required for the LR asymmetric looping. It was also found that the LR asymmetric cell rearrangement in the circular visceral muscle (CVM) is regulated by JNK signaling and required for the LR asymmetric looping of the AMG. Rac1, a Rho family small GTPase, augments JNK signaling in this process. These results also suggest that a basic mechanism for eliciting LR asymmetric gut looping may be conserved between vertebrates and invertebrates (Taniguchi, 2007).

This report, demonstrates that JNK signaling activity must be controlled properly in CVM cells for normal LR asymmetric development of the AMG. This idea is supported by the following observations. First, in the puc mutant, which has a hyperactivated JNK signal, the LR asymmetry of this organ is random, and the LR defects are rescued by puc expression in the trunk visceral mesoderm. Second, these LR defects are suppressed by the down-regulation of bsk, a Drosophila JNK homolog. Third, a stage-specific suppression of JNK signaling by the overexpression of puc results in laterality defects in the AMG. Importantly, however, there was no LR asymmetry in the expression pattern of the JNK signal or puc before or during the LR asymmetric development of this organ. It is therefore speculated that JNK signaling has a permissive role for LR asymmetric development of the AMG, rather than being an instructive cue for this process. For example, bilateral JNK signaling could have some influence on an instructive LR signaling molecule that has a LR differences in its activity or distribution. In this case, hyperactivation of JNK signaling may bring this instructive LR signal up or down across a threshold for the LR difference, triggering the LR asymmetric development of this organ (Taniguchi, 2007).

In Drosophila, several downstream targets of JNK signaling have been identified and studied extensively, and decapentaplegic and Delta are known downstream target genes of JNK signaling. However, the curret studies, including gene expression and genetic interaction analyses, demonstrate that these genes are unlikely to be involved in the JNK signal-driven LR asymmetric development of the AMG. In addition, previous report showed that the overexpression of Myo31DF inversed the laterality of the AMG. However, it was also found that puc did not influence the laterality defects induced by expressing Myo31DF, suggesting distinct roles for Myo31DF and JNK signaling in this process. In contrast to these observations, the expression of Fas3, which encodes an adhesion molecule localized to septate junctions, is significantly decreased in the CVM of the puc mutant. However, Fas3 is not required for the normal LR asymmetric development of the AMG. Therefore, it is presently unknown which target gene of JNK signaling is involved in the LR asymmetric development of the AMG (Taniguchi, 2007).

In contrast, this study demonstrates that the up-regulation of Rac1 activity in CVM cells results in LR defects of the AMG. In addition, Rac1 functions upstream of JNK in the CVM during LR asymmetric development of the AMG. Thus, the cytoskeletal rearrangement that is modulated by Rac1 upstream of JNK could be important in the LR asymmetric rearrangement of CVM cells. It is known that JNK activates head involution defective (hid) to induce apoptosis in and the clockwise rotation of the terminalia, which arises from the genital disc. In contrast to this organ's requirement for apoptosis for normal LR asymmetric development, this study found that the overexpression of p35, a potent suppressor of hid-dependent apoptosis, in CVM cells does not affect the LR asymmetric development of the AMG. In addition, the increased apoptosis of CVM cells was not observed in puc^GS16811 mutant embryos. These results suggest that, unlike its role in the genital disc, apoptosis is not involved in the LR asymmetric rearrangement of CVM cells, although JNK signaling plays essential roles in the LR asymmetric development of both organs (Taniguchi, 2007).

puc was found to be required for normal LR asymmetric development of the AMG at some time during stages 11 to 14. In addition, the LR asymmetric rearrangement of CVM cells is required for the normal laterality of the AMG. However, the LR asymmetric rearrangement of these cells occurs after stage 16. These results suggest that the down-regulation of JNK signaling by puc is required before, rather than during, the LR asymmetric cell rearrangement in the CVM. A possible explanation is that the down-regulation of JNK signaling is required to change the states of these cells to allow them to respond to the LR signal. It is speculated that a small LR bias at the initial stage, as found in CVM cells at late stage 15, is probably sufficient to introduce stereotypic LR asymmetry into the subsequent events of AMG morphogenesis. According to this model, the subsequent rotation of the AMG, which mostly occurs in the dorsoventral direction, augments the initial LR bias. Therefore, if the small bias is not introduced initially, the AMG shows nondirectional LR asymmetry, as was found in the puc mutant embryos (Taniguchi, 2007).

The Drosophila gut consists of two layers of cells, the epidermis and the visceral musculature. It was previously demonstrated that Myo31DF, which is essential for normal laterality of the hindgut, is required in the epithelium of this organ (Hozumi, 2006). This finding suggests that rearrangement of the epithelial cells is responsible for the LR asymmetric development of the embryonic hindgut. In contrast, for the normal LR asymmetric development of the AMG, the surrounding CVM cells play a crucial role (Taniguchi, 2007).

In zebrafish, the looping of the LR asymmetric gut is elicited as a secondary consequence of the LR asymmetric migration of the lateral plate mesoderm. The embryonic gut of this organism is located along the midline between the lateral plate mesoderm of each lateral half. The shape of the space between the left and right lateral plate mesodermal tissue becomes stereotypically LR asymmetrical, as a consequence of LR asymmetric changes in the configuration of these mesodermal tissues. Because the gut lies in the vacant space delineated by these tissues, the morphology of this organ also develops directional LR asymmetry. This study has demonstrated that the mesodermal tissue also plays an essential role in the LR asymmetric development of the Drosophila AMG. Although it is not known whether JNK signaling is involved in the LR asymmetric development of the lateral plate mesoderm in zebrafish, the roles of the mesodermal cells in the LR asymmetric development of the gut may be evolutionarily conserved between vertebrates and insects, at least to some extent (Taniguchi, 2007).

JNK signaling controls border cell cluster integrity and collective cell migration

Collective cell movement is a mechanism for invasion identified in many developmental events. Examples include the movement of lateral-line neurons in Zebrafish, cells in the inner blastocyst, and metastasis of epithelial tumors. One key model to study collective migration is the movement of border cell clusters in Drosophila. Drosophila egg chambers contain 15 nurse cells and a single oocyte surrounded by somatic follicle cells. At their anterior end, polar cells recruit several neighboring follicle cells to form the border cell cluster. By stage 9, and over 6 hr, border cells migrate as a cohort between nurse cells toward the oocyte. The specification and directionality of border cell movement are regulated by hormonal and signaling mechanisms. However, how border cells are held together while they migrate is not known. This study shows that negative-feedback loop controlling JNK activity regulates border cell cluster integrity. JNK signaling modulates contacts between border cells and between border cells and substratum to sustain collective migration by regulating several effectors including the polarity factor Bazooka and the cytoskeletal adaptor D-Paxillin. A role for the JNK pathway is anticipated in controlling collective cell movements in other morphogenetic and clinical models (Llense, 2008).

In an analysis of the mechanisms regulating the expression of puckered (puc), the gene encoding the Drosophila Jun N-terminal kinase (JNK) dual-specificity phosphatase (DSP) (Martin-Blanco, 1998) regulatory sequences (PG2) were uncovered directing its expression to border cells. PG2 expands across the first and second introns of puc, in which the puc^B48 insertion is located. This expression is also observed in puc enhancer (puc^B48) and protein trap lines (Llense, 2008).

JNKs represent a signaling hub with pivotal functions in cell proliferation, differentiation, and death. JNKs are inactivated by DSPs, and transcriptional induction of DSP expression is well documented as a negative-feedback mechanism. In Drosophila, this loop modulates JNK activity in processes such as epithelial expansion and overexpression of dominant-negative constructs relies on JNK signaling. Further, Puc overexpression leads to inhibition of JNK activity. Thus, Puc implements a negative-feedback loop in border cells (Llense, 2008).

Defects caused by the loss of JNK function in border cells included cluster dissociation and impaired motility. Instead of collectively following a leader cell, JNK-minus border cells autonomously disperse at the late step of migration, with most exhibiting long cellular extensions (LCEs) and actin-rich protrusions. JNK signaling does not affect polar cell specification or border cell recruitment (Llense, 2008).

Dissociation phenotypes are also observed in JNK-specific but not ERK-specific loss-of-function conditions for D-Fos, a major MAPK target, thereby ruling out potential interference via ERK. Indeed, reduced D-Fos suppresses border cell migration defects induced by elevated JNK activity (Llense, 2008).

Does JNK act in a linear pathway or does it target multiple independent effectors simultaneously to produce a multifaceted phenotype? Cells that migrate as part of a group cling firmly to each other while adhering transiently to the substrate. So, during migration, border cells show apicobasal polarity and remain attached to one another and to polar cells. Cell contacts are enriched in the adherens junctions (AJs) components, DE-Cadherin and Armadillo (β-Catenin). In electron microscopy (EM) preparations, border cells are tightly bound, whereas interfaces between border and nurse cells exhibit multiple interdigitations (Llense, 2008).

In JNK-minus conditions, namely after Puc overexpression or in bsk (JNK) clones, cell polarity is disrupted and only remnants of apical markers, such as Bazooka (Baz), are present. Adhesion is impaired, and DE-Cadherin and Armadillo are downregulated. Reduction of JNK activity also resulted in β-Integrin accumulation at ectopic actin-rich protrusions. These also accumulate MyoVI, consistent with its role in force generation. In summary, upon depletion of JNK activity, border cells lose apicobasal polarity and progress into a mesenchymal phenotype. Indeed, EM preparations show that border-border cell contacts are less tight than wild-type cell contacts and cell membranes detach from each other at multiple sites. The end result is a cluster with multiple leading edges and residual cell-cell contacts (Llense, 2008).

How does the JNK pathway become activated in border cells? Rho, Rac, and Cdc42 GTPases are potential candidates. Loss of Rac completely abolishes border cell migration. However, phenotypes for RhoA and Cdc42 expression of dominant-negative forms -- RhoA^DN and Cdc42^DN) closely resemble JNK-minus induced dissociation. Furthermore, in Cdc42^DN, polarity, cell contacts, and redistribution of substrate adhesion and motor markers are similarly affected. Most importantly, reporters of JNK activity such as Jun phosphorylation and the expression of the puc^B48 transgene are also downregulated. Null cdc42 MARCM clones display the same phenotype, although frequency and penetrancy were very low. Therefore, a role for other GTPases, such as RhoA, in JNK activation cannot be ruled out (Llense, 2008).

Border cell clusters deficient for Baz (BazRNAi) resemble JNK loss of function (which leads to Baz downregulation) and exhibit dissociation and downregulation of DE-Cadherin. Thus, Baz, a critical landmark of epithelial polarity, could serve as an effector for the control of border-border cell contacts. To test this, Baz was overexpressed in cells lacking JNK activity (or expressing Cdc42^DN); Baz was strongly rescued cluster integrity and DE-Cadherin expression (Llense, 2008).

Epithelial cells use a specialized repertoire of integrin receptors to mediate contacts and migration. However, border cells lacking β-Integrin were still able to adopt a leading migratory position, although the effect of complete removal of integrins from the cluster has not been reported (Llense, 2008).

Interestingly, β-Integrin antibodies reveal a rosette staining in border cell clusters that colocalize with AJ markers. Thus, β-Integrin could participate in the stabilization or strengthening of cell contacts, as shown for amnioserosa and larval epithelial cells in Drosophila, mammalian keratinocytes, and carcinoma cell clusters. Furthermore, β-Integrin, after JNK inactivation, strikingly accumulates at the front of LCEs suggesting a second function in cell invasiveness, as observed in leukocytes (Llense, 2008).

Direct evidence for β-Integrin involvement in border cell migration was obtained by RNAi in a sensitized JNK-minus condition. The expression of β-Integrin dsRNAs in border cells reduced β-Integrin levels but did not cause migration or integrity defects. However, in the presence of Puc, β-Integrin RNAi led to a strong enhancement of cluster dissociation and prevented the full extension of LCEs, which become mostly blunted. Moreover, an adhesion dominant negative (diβ) integrin chimera showed weak, but reproducible, dissociation phenotypes. Thus, β-Integrin turns out to participate in, first, the stabilization of border-border cell contacts and, second, the promotion of LCEs extension. The integrin countereceptors that facilitate border cell attachment and invasiveness are not yet known (Llense, 2008).

D-Paxillin was present in border cell contacts but was downregulated in JNK-minus conditions. Genomic-profiling analyses of JNK mutants suggests a transcriptional control of D-Paxillin expression. However, other options, such as subcellular relocation after phosphorylation, could also explain why D-Paxillin may be absent from JNK-minus border cells. Expression in border cells of two different D-Paxillin dsRNA lines was found to result in JNK loss-of-function-like dissociation, DE-Cadherin downregulation and β-Integrin accumulation at LCEs. Expression of a Talin RNAi line does not produce any migration phenotype, although it impairs follicle epithelia integrity (Llense, 2008).

In migratory leukocytes, PKA-mediated integrin phosphorylation prevents Paxillin accumulation at the leading front. Paxillin-integrin interactions in lateral positions lead to the inhibition of Rac, whose activation is thus spatially limited to the leading edge where it induces lamellipodia. Consequently, D-Paxillin might stabilize β-Integrin in border-border cell contacts. Its absence, in JNK-minus conditions, would lead in lateral and trailing cells to Rac activation, dissociation of border-border cell contacts, and extension of β-Integrin-rich ectopic lamellipodia. Indeed, the PKA-RII subunit is expressed in border cells, and border cells mutant for PKA show migration defects (Llense, 2008).

Interestingly, D-Paxillin overexpression rescued the border cell defect resulting from loss of JNK activity (or expression of Cdc42^DN). DE-Cadherin relocated to border-border cell contacts, and β-Integrin expression was partially eliminated from residual LCEs. D-Paxillin overexpression alone had no effects (Llense, 2008).

It was further asked whether the control of cell polarity and cytoskeletal adaptor proteins by JNK were related. Paxillin expression was strongly reduced in baz mutant conditions, whereas Baz expression was only slightly affected by interference in Paxillin expression (Llense, 2008).

JNK signaling regulates border cells clustering by controlling at least two key elements, cell polarity (Baz) and cytoskeletal adaptor proteins (D-Paxillin), and as a consequence cell-cell contacts and cell-substrate attachments. Interestingly, the overexpression of Hindsight (Hnt), a target and negative regulator of JNK, results in similar defects to those caused by inhibition of JNK. Because re-expression of a variety of proteins (Baz, D-Paxillin, DE-Cadherin, and Armadillo) can rescue the dissociation phenotype and given that each time rescue is achieved, DE-Cadherin and Armadillo expression are restored, a plausible explanation for the effects observed with JNK-minus and Hnt overexpression is that there is an overall loss of multiple cell-cell adhesion complexes. The restoration of any of them would provide sufficient cell-cell adhesion to enable the cluster to move as a collective (Llense, 2008).

The individual migratory abilities of JNK-minus border cells could be partially explained by the observed β-Integrin relocalization to LCEs (border-nurse cell contacts). Alternatively, border cells could have lost their capacity to respond to positional gradients leading to random outward movements. Border cells use PVF and EGF to guide their migration. Blocking PVR and EGFR does not reduce the ability of border cells to extend protrusions but abolishes their directionality, with protrusions now extending in all directions. However, in these conditions, border cell clusters do not dissociate, thereby ruling out the possibility that dissociation in JNK mutants is due only to loss of directional guidance. A directionality index (DI) can be calculated. A DI of 0 indicates equal numbers of protrusions extending forward and backward. A DI of 1 indicates that cells only extend protrusions in the direction of migration. This study found a DI of 0.59 for wild-type clusters. In the absence of JNK, however, clusters show a DI ranging from -0.2 to 0, suggesting that JNK-minus border cells are blind to positional cues. This fact accounts for recently described synergistic effects of JNK and PVR signaling on border cells (Llense, 2008).

This model makes a significant prediction: JNK hyperactivation should increase adhesiveness and eventually block migration. Accordingly, ut was observed that the overexpresssion of a constitutively active form of Hep, the overexpresssion of a constitutively active form of Misshapen, or loss of function clones of puc resulted in nonmigratory and strongly compacted clusters. Occasionally, the death of a number of border cells was observed (Llense, 2008).

So far, the molecular and cellular study of collective versus individual migration both in developmental and cancer models has mainly focused on the analysis of structural elements. The identification of the JNK cascade as a key determinant of migratory responses in border cells could have an important impact in the understanding of collective movements. Border cell migration could serve as a good model for studying migratory transitions, thus impacting on the understanding of cancer metastasis and invasiveness, during which so little is known about the signaling mechanisms controlling migratory behavior (Llense, 2008).

Raw mediates antagonism of AP-1 activity in Drosophila

High baselines of transcription factor activities represent fundamental obstacles to regulated signaling. This study shows that in Drosophila, quenching of basal activator protein 1 (AP-1) transcription factor activity serves as a prerequisite to its tight spatial and temporal control by the JNK (Jun N-terminal kinase) signaling cascade. These studies indicate that the novel raw gene product is required to limit AP-1 activity to leading edge epidermal cells during embryonic dorsal closure. In addition, evidence is provided that the epidermis has a Basket JNK-independent capacity to activate AP-1 targets and that raw function is required broadly throughout the epidermis to antagonize this activity. Finally, mechanistic studies of the three dorsal-open group genes [raw, ribbon (rib), and puckered (puc)] indicate that these gene products provide at least two tiers of JNK/AP-1 regulation. In addition to Puckered phosphatase function in leading edge epidermal cells as a negative-feedback regulator of JNK signaling, the three dorsal-open group gene products (Raw, Ribbon, and Puckered) are required more broadly in the dorsolateral epidermis to quench a basal, signaling-independent activity of the AP-1 transcription factor (Bates, 2008).

The initial molecular and genetic studies of the dorsal-open mutant raw revealed it to encode a widely expressed and novel gene product, required for the restriction of JNK/AP-1 activity to LE epidermal cells (Byars, 1999). The Raw protein sequence yielded no insights into its mechanism of function as the Raw sequence harbors none of the canonical motifs that are associated with nuclear localization, phosphorylation, membrane insertion, or protein secretion. Mechanistic studies of a novel protein can be challenging, but this study reports use of a variety of genetic strategies to probe Raw function and test models of AP-1 silencing. In particular (1) the epistatic relationship of raw to genes encoding well-characterized JNK-signaling components was assessed, (2) genes, which have designated the raw group, have been assessed that share an array of loss-of-function phenotypes, (3) the interaction phenotypes among the raw-group loci were determined, and (4) raw transgenics were generated, that were utilized to probe sites of Raw function. These analyses reveal that raw belongs to a small set of dorsal-open group genes that encode JNK/AP-1 pathway antagonists. The characterization of raw, and the raw group more generally, has led to a new appreciation of wide-ranging competence for AP-1 activity in early Drosophila embryos. As signal activation is critical for proper development, so also is its silencing (Bates, 2008).

The current study shows that although raw functions upstream of Jra as an AP-1 antagonist, its action is independent of the bsk-encoded kinase that is required to activate AP-1 activity in LE cells during closure. In addition, raw is required broadly in the epidermis to effect normal dorsal closure. Overall, these studies expose the importance of epidermal AP-1 silencing during embryogenesis and lead to an extension of existing models for dorsal closure, which have largely confined their focus to mechanisms of JNK/AP-1 activation in LE cells. In particular, the data indicate that Raw and the other raw-group gene products (Puckered and Ribbon) function to silence Basket JNK-independent AP-1 activity in the embryonic dorsolateral epidermis. AP-1 silencing, via the combined actions of the raw-group gene products, essentially wipes the epidermal slate clean and primes the system for activation via a still unidentified deterministic signal that acts only in LE cells (Bates, 2008).

The AP-1 abnormality in raw-group mutant embryos has not yet been molecularly defined. Previous studies provide compelling evidence that AP-1 overexpression in Drosophila embryos is not sufficient to disrupt either dorsal closure or development more generally. It seems unlikely, therefore, that elevated levels of the AP-1 transcription factor in raw-group mutants simply override a requirement for kinase activation in initiating an AP-1-dependent program of gene expression. Instead, it is speculated that AP-1 is aberrantly modified in raw-group mutant embryos. It might be that AP-1 escapes inactivation in mutants; either alternatively or additionally, AP-1 in mutants may be inappropriately activated via phosphorylation. In addition to Basket JNK, there are four other Drosophila MAP Kinases (p38a, p38b, Mpk2, and Rolled) that might provide dysregulated kinase activity in mutants. Consistent with this idea is the observation that the oogenesis phenotypes associated with raw (and puc) ectopic expression and mutation have considerable similarity with gain- and loss-of-function phenotypes associated with mutations in the p38 pathway that is required in the germ line for proper oogenesis. Finally, a kinase-dependent activation model for epidermal Jun provides the most parsimonious explanation for ectopic epidermal signaling observed in puc MPK-deficient embryos. From the perspective of regulated signaling more generally, however, lowering an AP-1 activity baseline in wild-type embryos will (1) provide a means for the clean on/off regulation of JNK/AP-1 that has been predicted in computer simulations and (2) make a less strenuous demand on the input activating signal (Bates, 2008).

The discovery that null alleles of raw and puc interact, with double mutants exhibiting an embryonic lethal phenotype distinct from their shared loss-of-function null phenotypes, revealed the independent contributions of raw and puc to embryogenesis, presumably through their effects on AP-1 antagonism. Drosophila overexpression studies have previously implicated several pathways in the parallel control of AP-1 activity, but this analysis represents the first direct demonstration of physiologically relevant, parallel regulatory pathways (Bates, 2008).

The genetic interaction that was documented between null alleles of raw and puc contrasts with the lack of a detectable interaction between null alleles of raw and rib. Moreover, the observation that raw and rib hypomorphs interact genetically during dorsal closure is consistent with previously published data, as well as with findings documenting (1) raw/rib interactions in several other epithelial tissues, including the nervous system, salivary gland, trachea, and gut (Blake, 1998; Blake, 1999) and (2) overlapping raw and rib expression patterns in Drosophila embryos (Byars, 1999). Together, results from these genetic and molecular studies point to roles for raw and rib in a single, previously unrecognized puc-independent AP-1 inactivation system (Bates, 2008).

In addition to providing evidence for raw-mediated global silencing of AP-1, this study underscores a simultaneous requirement for a biologically appropriate activator of JNK/AP-1 signaling. In this regard, expression of raw in LE cells failed to rescue raw-dependent defects in dorsal closure. Even more notable, however, was the observation that overexpression of raw⁺ in wild-type embryos, and in wild-type LE cells in particular, had no detrimental effects on embryonic development and dorsal closure. From a signaling perspective this result indicates that JNK-dependent AP-1 can be activated despite expression of the wild-type raw gene product, and thus Raw does not function as a binary switch for signaling. Although it is formally possible that LE expression of raw was initiated too late to disrupt JNK/AP-1 signaling and dorsal closure in the LE-gal4/UAS-raw⁺ transgenics, this interpretation is not favored since the LE-GAL4 driver used in this study has been shown previously to (1) be an effective driver of at least one gene that is required in LE epidermal cells for closure and (2) drive expression of a lacZ reporter in LE cells during dorsal closure (Bates, 2008).

The finding that raw expression in LE cells is not sufficient to inactivate AP-1 activity in a cell-autonomous fashion is consistent with models for independent, developmentally regulated triggers of JNK signaling. Indeed, there is abundant experimental support for developmentally regulated activation of JNK signaling in LE cells. JNK/AP-1 activation likely follows an amalgamation of signals, both from the amnioserosa and the epidermis, both in the form of cytoskeletal components and signaling molecules. Among the best candidates with postulated roles in JNK/AP-1 activation are small GTPases, nonreceptor tyrosine kinases, and integrins. Thus, despite the broad epidermal competence for AP-1 signaling that has been shown in this work, the activation signal is itself limited to only LE cells and functions via an unknown mechanism. Importantly, AP-1 antagonism by raw cannot override its signal-dependent activation in the LE (Bates, 2008).

dpp, when expressed pan-epidermally, leads to a raw-like phenotype: embryonic lethality associated with ventral cuticular defects. In a direct assessment of equivalence of raw loss-of-function and dpp gain-of-function ventral cuticular phenotypes, whether pan-epidermal expression of brinker (brk) can rescue raw-dependent defects in the ventral cuticle was tested. The Dpp signaling modifier Brinker functions by negatively regulating dpp target genes (Bates, 2008).

This study found that although brk is normally expressed in nonoverlapping lateral and ventral domains of the embryonic epidermis, it is undetectable in the epidermis of embryos homozygous for a null allele of raw. It was also found that although brk⁺ fails to rescue raw-dependent defects in dorsal closure, it does rescue raw-dependent defects in the ventral cuticle. Together, these data point to an important role for dpp, brk, and/or their target genes in development of the ventral epidermis (Bates, 2008).

What cannot be discerned from these studies is (1) how the nonoverlapping epidermal domains of dpp and brk are established and maintained and (2) if and how epidermal dpp and brk interact during normal embryonic development. In this regard, a previous finding that LE dpp is not autoregulatory makes it unlikely that brk functions in direct fashion to set the LE dpp expression boundary. Even more significant is the finding that cuticles derived from Jra raw double mutants exhibit defects in dorsal closure, but not ventral cuticular patterning (BYARS, 1999). Indeed, these data highlight the requirement for functional Jun in generating ventral cuticular defects in raw mutant embryos. Taken together then, these data suggest that the effects of JNK/AP-1-activated dpp in the dorsal epidermis of raw mutant embryos are far reaching, extending even to the most ventral regions of the embryo (Bates, 2008).

Having established a dependence upon Jun for raw-dependent ventral cuticular defects, it is postulated that the absence of brk in raw mutant embryos is a direct consequence of ectopic JNK/AP-1 activity in the dorsal epidermis of these mutants. It is suspected that ectopic JNK/AP-1 activity leads secondarily to ectopic dpp activity, and that in its turn ectopic dpp activity leads finally to brk repression. An alternative view, that raw might have dual regulatory roles in the epidermis, seems less likely although it is not absolutely excluded by this strictly genetic analysis. In this regard, in addition to its function as a JNK/AP-1 antagonist in the embryonic dorsal epidermis, raw might function independently as a trigger of brk expression in the ventral epidermis. Clearly, the mechanism of raw function and the relationship of dpp to brk in eliciting properly formed ventral cuticle warrant further investigation (Bates, 2008).

In Drosophila, as in all animals, signaling pathways are finely regulated at several levels. Although there are multiple tiers of regulation operating on the JNK/AP-1 signaling cascade, surprisingly little of the regulation of this pathway is known. This study of the functions and interactions of a subset of dorsal-open group genes (raw, rib, and puc) has shed some additional light on both old (puc-mediated) and new (raw/rib-mediated) mechanisms of JNK/AP-1 antagonism. These data indicate that Raw functions to silence Basket JNK-independent AP-1-mediated transcription and to set the stage for JNK-dependent regulation of transcription. The suggestion that spatial restriction of the JNK/AP-1 signal requires antagonists, as well as activators, is not without precedent in other signaling systems. Many signaling pathways have already been shown to be multilayered and to depend heavily on negative regulation to terminate developmental events, and/or control both the distance and speed that a signal can move (e.g., Nodal). In addition, and as was suggested is the case for the Drosophila JNK/AP-1 pathway, reducing basal levels of a signaling pathway can augment the effects of its signaling responses (e.g., Hedgehog and Lef1) (Bates, 2008).

Finally, given the numerous associations of improper JNK/AP-1 activity with human disease, it seems apparent that many cell types have the capacity to signal via the JNK/AP-1 pathway. Presumably, this capacity is diminished (and then tightly regulated) during normal vertebrate development and aging. Viewed from this perspective, characterization of Raw as an essential AP-1 antagonist establishes a clear basis for future studies of AP-1 regulation (Bates, 2008).

Invasive and indigenous microbiota impact intestinal stem cell activity through JAK-STAT and JNK pathways in Drosophila

Gut homeostasis is controlled by both immune and developmental mechanisms, and its disruption can lead to inflammatory disorders or cancerous lesions of the intestine. While the impact of bacteria on the mucosal immune system is beginning to be precisely understood, little is known about the effects of bacteria on gut epithelium renewal. This study addressed how both infectious and indigenous bacteria modulate stem cell activity in Drosophila. The increased epithelium renewal observed upon some bacterial infections is a consequence of the oxidative burst, a major defense of the Drosophila gut. Additionally, evidence is provided that the JAK-STAT and JNK pathways are both required for bacteria-induced stem cell proliferation. Similarly, it was demonstrated that indigenous gut microbiota activate the same, albeit reduced, program at basal levels. Altered control of gut microbiota in immune-deficient or aged flies correlates with increased epithelium renewal. Finally, it was shown that epithelium renewal is an essential component of Drosophila defense against oral bacterial infection. Altogether, these results indicate that gut homeostasis is achieved by a complex interregulation of the immune response, gut microbiota, and stem cell activity (Buchon, 2009).

The JAK-STAT and JNK signaling pathways are required to maintain gut homeostasis upon exposure to a broad range of bacteria. In normal conditions, low levels of the indigenous gut microbiota and transient environmental microbes maintain a basal level of epithelium renewal. The increase in gut microbes in old or Imd-deficient flies is associated with a chronic activation of the JNK and JAK-STAT pathways, leading to an increase in intestinal stem cells (ISC) proliferation and gut disorganization. The impact of pathogenic bacteria can have different outcomes on gut homeostasis, depending on the degree of damage they inflict on the host. Damage to the gut caused by infection with E. carotovora is compensated for by an increase in epithelium renewal. Infection with a high dose of P. entomophila disrupts the homeostasis normally maintained by epithelium renewal and damage is not repaired, contributing to the death of the fly (Buchon, 2009).

Previous studies have shown that the NADPH oxidase Duox plays an essential role in Drosophila gut immunity by generating microbicidal effectors such as ROS to eliminate both invasive and dietary microbes. Ecc15 is a potent activator of Duox, which in turn is important in the clearance of this bacterium. This oxidative burst is coordinated with the induction of many genes involved in ROS detoxification upon Ecc15 ingestion. This study provides evidence that the observed increase in epithelium renewal upon Ecc15 infection is a compensatory mechanism that repairs the damage inflicted to the gut by this oxidative burst. This is supported by the observation that reducing ROS levels by either the ingestion of antioxidants or silencing the Duox gene reduces epithelium renewal. Although ISC proliferation could be directly triggered by ROS, it is more likely a consequence of signals produced by stressed enterocytes. A number of data support this hypothesis: (1) Ingestion of corrosive agents can also induce ISC proliferation, and (2) physical injury is sufficient to induce local activation of the cytokine Upd3, which promotes epithelium renewal. Interestingly, a significant increase in epithelium renewal was observed in Duox RNAi flies at late time points following infection, correlating with damage attributed to the proliferation of Ecc15 in the guts of Duox-deficient flies. While the increase in epithelium renewal observed with Ecc15 is clearly linked to the damage induced by the host immune response, it is likely that effects on epithelium renewal by other pathogens could be more direct and mediated by virulence factors, such as the production of cytolytic toxins (Buchon, 2009).

The data indicate that the JAK-STAT and JNK pathways synergize to promote ISC proliferation and epithelium renewal in response to the damage induced by infection. The JAK-STAT pathway is implicated in the regulation of stem cells in multiple tissues and is proposed to be a common regulator of stem cell proliferation. The data extend this observation by showing that the JAK-STAT pathway is also involved in ISC activation upon bacterial infection. The cytokine Upd3 is produced locally by damaged enterocytes and subsequently stimulates the JAK-STAT pathway in ISCs to promote their proliferation. The results globally agree with a recent study showing that the JAK-STAT pathway is involved in ISC proliferation upon infection with a low dose of P. entomophila (Jiang, 2009). This work and the current study clearly demonstrate that the JAK-STAT pathway adjusts the level of epithelium renewal to ensure proper tissue homeostasis by linking enterocyte damage to ISC proliferation. The study by Jiang also uncovered an additional role of this pathway in the differentiation of enteroblasts during basal gut epithelium turnover. The implication of the JAK-STAT pathway in differentiation could explain the accumulation of the small-nucleated escargot-positive cells observed in the gut of flies with reduced JAK-STAT signaling in ISCs. The JAK-STAT pathway was also shown previously to control the expression of some antimicrobial peptides such as Drosomycin 3 (Dro3). Therefore, the JAK-STAT pathway has a dual role in the gut upon infection, controlling both the immune response and epithelium renewal (Buchon, 2009).

The data show that the lack of JNK pathway activity in ISCs results in the loss of ISCs in guts infected with Ecc15, thus preventing epithelium renewal. The findings are consistent with the attributed function of JNK at the center of a signal transduction network that coordinates the induction of protective genes in response to oxidative challenge. This cytoprotective role against ROS would protect ISCs from the oxidative burst induced upon Ecc15 infection, explaining why ISCs die by apoptosis when JNK activity is reduced. It is likely that JNK signaling is required not only to protect ISCs from oxidative stress, but also to induce stem cell proliferation to replace damaged differentiated cells. This is supported by the observation that overexpression of the JNKK Hep in ISCs is sufficient to trigger an epithelium renewal in the absence of infection. In addition, increased JNK activity in ISCs of old flies has been linked to hyperproliferative states and age-related deterioration of the intestinal epithelium. This study shows that JNK signaling is also required for epithelium renewal upon Ecc15 infection. Thus, infection with Ecc15 recapitulates in an accelerated time frame the impacts of increased stress observed in guts of aging flies (Buchon, 2009).

The inhibition of the dJun transcription factor in ISCs leads to a loss of stem cells in the absence of infection, suggesting that this transcription factor plays a critical role in ISC maintenance in the gut. There is no definitive explanation for why the dJun-IR construct behaves differently than the basket and hep-IR constructs. It is speculated that this could be due to (1) differences in the basal activity of the JNK pathway, which would be blocked only with the dJun-IR that targets a terminal component of the pathway; (2) effects of Jun in ISCs independent of the JNK pathway; or (3) side effects of the dJun-IR construct (Buchon, 2009).

In contrast to the requirement of the JNK pathway upon Ecc15 infection, it has been reported that oral ingestion with a low dose of P. entomophila still induced mitosis in the JNK-defective mutant hep¹. In agreement, this study found that inhibiting the JNK pathway in ISCs did not block the induction of epithelium renewal by a low dose of P. entomophila. This difference in the requirement of the JNK pathway may be explained by the nature of these two pathogens. Whereas Ecc15 damages the gut through an oxidative burst that activates the JNK pathway, the stimulation of epithelium renewal by P. entomophila could be due to a more direct effect of this bacterium on the gut. Altogether, this work points to an essential role of the JAK-STAT pathway in modulation of epithelium renewal activity, while the role of JNK may be dependent on the infectious agent and any associated oxidative stress. While it is known that the JNK pathway is activated by a variety of environmental challenges including ROS, the precise mechanism of activation of this pathway has not been elucidated. Similarly, the molecular basis of upd3 induction in damaged enterocytes is not known. Future work should decipher the nature of the signals that activate these pathways in both ISCs and enterocytes, as well as the possible cross-talk between the JNK and JAK-STAT pathways in ISC control (Buchon, 2009).

The observation that flies unable to renew their gut epithelium eventually succumb to Ecc15 infection highlights the importance of this process in the gut immune response. It is striking that defects in epithelium renewal are more detrimental to host survival than deficiency in the Imd pathway, even though this pathway controls most of the intestinal immune-regulated genes induced by Ecc15. The results are in agreement with a previous study indicating that, in the Drosophila gastrointestinal tract, the Imd-dependent immune response is normally dispensable to most transient bacteria, but is provisionally crucial in the event that the host encounters ROS-resistant microbes. However, this study demonstrates that efficient and rapid clearance of bacteria in the gut by Duox is possible only when coordinated with epithelium renewal to repair damage caused by ROS. This finely tuned balance between bacterial elimination by Duox activity and gut resistance to collateral damage induced by ROS is likely the reason why flies normally survive infection by Ecc15. Yet, this calibration also exposes a vulnerability that could easily be manipulated or subverted by other pathogens. Along this line, this work also exposes the range of impact different bacteria can have on stem cell activation. It was observed that infection with high doses of P. entomophila led to a loss of gut integrity, including the loss of stem cells. Moreover, the ability of P. entomophila to disrupt epithelium renewal correlates with damage to the gut and the death of the host. Since both JNK and JAK-STAT pathways are activated upon infection with P. entomophila, this suggests that this bacterium activates the appropriate pathways necessary to repair the gut, but ISCs are unable to respond accordingly. Interestingly, a completely avirulent P. entomophila mutant (gacA) does not persist in the gut and does not induce epithelium renewal. In contrast, an attenuated mutant (aprA) somewhat restores epithelium renewal. These observations, along with the dose response analysis using P. entomophila and corrosive agents, suggest that the virulence factors of this entomopathogen disrupt epithelium renewal through excessive damage to the gut. Of note, recent studies suggest that both Helicobacter pylori and Shigella flexneri, two bacterial pathogens of the human digestive tract, interfere with epithelium renewal to exert their pathological effects. This suggests that epithelium renewal could be a common target for bacteria that infect through the gut. In this respect, the host defense to oral bacterial infection could be considered as a bimodular response, composed of both immune and homeostatic processes that require strict coordination. Disruption of either process results in the failure to resolve the infection and impedes the return to homeostasis (Buchon, 2009).

In contrast to the acute invasion by pathogenic bacteria, indigenous gut microbiota are in constant association with the gut epithelium, and thus may impact gut homeostasis. Using axenically raised flies, it was established that indigenous microbiota stimulate a basal level of epithelium renewal that correlates with the level of activation of the JAK-STAT and JNK pathways. This raises the possibility that both indigenous and invasive bacteria, such as Ecc15, are capable of triggering epithelium renewal by the same process. Additionally, the data support a novel homeostatic mechanism in which the density of indigenous bacteria is coupled to the level of epithelium renewal. This is the first report that gut microbiota affect stem cell activation and epithelium renewal, concepts proposed previously in mammalian systems but never fully demonstrated. This also implies that variations in the level of epithelium renewal observed in different laboratory contexts could actually be due to impacts from gut microbes (Buchon, 2009).

Importantly, in this context, it was shown that lack of indigenous microbiota reverts most age-related deterioration of the gut. Aging of the gut is usually marked by both hyperproliferation of ISCs and differentiation defaults that lead to disorganization of the gut epithelium. These alterations have been shown to be associated with activation of the PDGF- and VEGF-related factor 2 (Pvf2)/Pvr and JNK signaling pathways directly in ISCs. Accordingly, inhibition of the JNK pathway in ISCs fully reverts the epithelium alterations that occur with aging. This raises the possibility that gut microbiota could exert their effect through prolonged activation of the JNK pathway. Interestingly, immune-deficient flies, lacking the Imd pathway, also display hyperproliferative guts and have higher basal levels of activation of the JNK and JAK-STAT pathways. The observation that these flies also harbor higher numbers of indigenous bacteria further supports a model in which failure to control gut microbiota leads to an imbalance in gut epithelium turnover. Future work should analyze the mechanisms by which gut microbiota affect epithelium renewal and whether this is due to a direct impact of bacteria on the gut or is mediated indirectly through changes in fly physiology. Moreover, the correlation between higher numbers of indigenous bacteria and increased disorganization of the gut upon aging in flies lacking the Imd pathway raises the possibility that a main function of this pathway is to control gut microbiota. This is in agreement with concepts emerging in mammals that support an essential role of the gut immune response in maintaining the beneficial nature of the host-microbiota association. This function also parallels the theory of 'controlled inflammation' described in mammals, where a low level of immune activation is proposed to maintain gut barrier integrity (Buchon, 2009).

In conclusion, this study unravels some of the complex interconnections between the immune response, invasive and indigenous microbiota, and stem cell homeostasis in the gut of Drosophila. Based on the evolutionary conservation of transduction pathways such as JNK and JAK-STAT between Drosophila and mammals, it is likely that similar processes occur in the gut of mammals during infection. Interestingly, stimulation of stem cell activity by invasive bacteria is proposed to favor the development of hyperproliferative states found in precancerous lesions. Thus, Drosophila may provide a more accessible model to elucidate host mechanisms to maintain homeostasis and the impact of bacteria on this process (Buchon, 2009).

Scarface, a secreted serine protease-like protein, regulates polarized localization of laminin A at the basement membrane of the Drosophila embryo acting as a JNK antagonist

Cell-matrix interactions brought about by the activity of integrins and laminins maintain the polarized architecture of epithelia and mediate morphogenetic interactions between apposing tissues. Although the polarized localization of laminins at the basement membrane is a crucial step in these processes, little is known about how this polarized distribution is achieved. This study analysed the role of the secreted serine protease-like protein Scarface in germ-band retraction and dorsal closure -- morphogenetic processes that rely on the activity of integrins and laminins. Evidence that scarface is regulated by c-Jun amino-terminal kinase and that scarface mutant embryos show defects in these morphogenetic processes. Anomalous accumulation of laminin A on the apical surface of epithelial cells was observed in these embryos before a loss of epithelial polarity was induced. It is proposed that Scarface has a key role in regulating the polarized localization of laminin A in this developmental context (Sorrosal, 2010).

During germ band retraction (GBR), the tail end of the germ band, or embryo proper, interacts with the amnioserosa (AS), an epithelium of large flat cells that does not contribute to the larva, and moves to its final posterior position. After GBR, the ectoderm has a gap on its dorsal side that is occupied by the amnioserosa (AS). The dorsal-most cells of the ectoderm, on both sides of the embryo, are in contact with the AS and are called leading edge (LE) cells. During DC, LE cells direct the movement of the epidermal sheets migrating dorsally over the apical side of the AS until they meet and fuse at the dorsal midline. Attention of this study centered on scarf on the basis of its embryonic expression pattern. Two piggyBac insertions, scarf ^PBss(GFP) and scarf ^M13.M2(lacZ), were expressed at a high level in LE cells during GBR and DC and at a low level in AS cells. The expression of scarf was confirmed by in situ hybridization. It was also expressed in the head and tail regions, and in the ventral ectoderm in a segmental pattern (Sorrosal, 2010).

The JNK pathway is activated in LE cells and leads to the expression of the signalling molecule Dpp and the phosphatase Puckered (Puc). The scarf gene was expressed in the same cells as puc and dpp at the LE. Reduced JNK - Basket (Bsk) in Drosophila - or expression of a dominant-negative version of Bsk or Puc, which mediates a feedback loop repressing JNK activity, led to the loss of scarf expression in LE cells. Loss of puc activity, which leads to increased levels of JNK activity, or expression of an activated version of JNK -activating kinase (Hemipterous in Drosophila), led to the expansion of the scarf expression domain throughout the lateral ectoderm. Dorsal cells had a stronger response to increased JNK, suggesting that JNK requires the activity of another factor expressed in this region to induce scarf. As stated, the JNK cascade drives the expression of the secreted molecule Dpp. In embryos mutant for the Dpp receptor Thickveins, scarf expression at the LE was not lost and ectopic activation of the Dpp pathway did not induce the ectopic expression of scarf. These results indicate that scarf expression is induced by JNK in LE cells (Sorrosal, 2010).

Homozygosis or trans-heterozygosis for the piggyBac insertions scarf ^PBss and scarf ^M13.M2 caused semi-lethality and survivors had scars on the head, phenotypes that were reverted by precise excision of the transposon elements. As embryos showed no obvious cuticle phenotype, stronger alleles of scarf were generated by imprecise excision of a P element located in the third intron of scarf (scarf ^KG05129). One allele (scarf ^Δ1.5) was isolated that led to the loss of scarf messenger RNA expression. This is an embryonic lethal allele and trans-heterozygous combinations of scarf ^Δ1.5 or Df(2R)nap¹⁴, a deficiency that uncovers the scarf locus, with scarf ^PBss or scarf ^M13.M2 were semi-lethal and resulted in scars on the head. Homozygous animals for scarf ^Δ1.5 or Df(2R)nap¹⁴ showed similar phenotypes. Their larval cuticles were either dorsally wrinkled or presented a dorsal hole or a U-shaped phenotype, which is suggestive of failures in GBR and DC. A high proportion of embryos showed undifferentiated cuticles. Ubiquitous expression of scarf largely rescued the scarf ^Δ1.5 phenotype (Sorrosal, 2010).

Time-lapse recordings indicated that these embryos were delayed in development and showed a failure in GBR. Frequently, the attachment of the AS to the tail end of the germ band was compromised and the germ band did not retract. In embryos that were able to retract, DC started and LE cells began to direct the movement of the epidermal sheets migrating dorsally over the apical side of the AS. However, either the AS detached from the neighbouring epidermal sheet or the epidermal sheets met and fused at the dorsal midline but the dorsal epidermis reopened. The requirement of scarf in DC was analyzed in greater detail in fixed staged embryos. During the initial steps of DC, lateral ectodermal cells start to elongate dorsally and an actin cable at the LE is formed, which helps to generate a linear fence at the interface between LE and AS cells. In scarf ^Δ1.5 embryos, elongation of the lateral ectoderm was compromised frequently, the actin cable was not formed properly, the interface between LE and AS cells became irregular, and eventually rips appeared at the AS-LE interface. The elongation of lateral ectodermal cells and adhesion between AS and LE cells are mediated by the activity of Dpp and JNK activity controls the polymerization of actin into a cable at the LE. In embryos lacking scarf, the activity of the Dpp and JNK signalling pathways was not affected. Thus, Scarf has a role in DC without affecting the activity levels of these pathways (Sorrosal, 2010).

Integrins and laminins have a fundamental role in GBR and DC by mediating interactions between AS cells and neighbouring cell populations. As defects were observed in these processes on scarf depletion and a reduction in scarf levels increased the frequency of cuticle phenotypes caused by depletion of the β-integrin position-specific (βPS) subunit, the contribution of Scarf to the regulation of the expression levels and subcellular localization of these proteins was examined. Cross-sectional views of properly staged wild-type and scarf ^Δ1.5 embryos stained for βPS integrin revealed similar levels and localization of integrins. The JNK signalling regulates the expression of the βPS integrin subunit during DC. Although ectopic activation of JNK induced increased expression of βPS integrin, ectopic expression of scarf did not exert this effect. The expression of LanA, one of the two existing fly α-laminins. In wild-type embryos, high levels of LanA were localized at the BM of AS cells. Interestingly, scarf embryos revealed strong defects in the polarized distribution of LanA. It was localized on the apical side of the AS epithelium and its protein levels were largely reduced in the BM. Ubiquitous expression of scarf largely rescued the levels of LanA at the BM. Similar defects were observed in the polarized distribution of LanA in the lateral ectoderm of scarf mutant embryos, and these defects were also rescued largely by the ubiquitous expression of scarf. Apico-basal polarity of AS cells, visualized by the basal localization of βPS integrin and the localization of E-cadherin (E-cad) at the adherens junctions, was not affected in hypomorphic scarf ^PBss and scarf ^M13.M2 embryos and in some scarf ^Δ1.5 embryos even though LanA was localized on the apical side. However, scarf depletion was frequently found to cause defects in the apico-basal polarity of AS cells and this was accompanied by multilayering of the AS epithelium. These observations suggest that before inducing a loss of epithelial integrity, the absence of Scarf caused aberrant localization of LanA on the apical side of the AS epithelium and reduced LanA in the BM, without affecting the distribution of apical and baso-lateral proteins. As integrins are involved in maintaining epithelial polarity, these results suggest that the observed loss of cell polarity is a consequence of reduced integrin-mediated adhesion to the BM. Consistent with this view, defects in the attachment of the AS cells to the underlying yolk cell were observed frequently (Sorrosal, 2010).

Although the defects observed in scarf ^Δ1.5 embryos resemble those caused by βPS integrin depletion, only defects in the attachment of the AS with the underlying yolk cells are found in lanA mutant embryos. Mutations in wing blister (wb), the only other fly α-laminin, cause defects in GBR and in the attachment between AS cells and the posterior germ band. These data suggest that LanA and Wb have a redundant function and that Scarf most probably promotes BM localization of not only LanA but also Wb. Consistent with the proposed redundancy, halving the dose of lanA or wb increased the frequency of the βPS integrin loss-of-function cuticle phenotype. The expression of Wb protein in AS cells using the available antibodies and the role of Scarf in the polarized distribution of other BM proteins, such as Perlecan or Collagen IV, were not addressed as these two proteins were not expressed in AS cells (Sorrosal, 2010).

These results indicate that Scarf depletion causes defects in the BM localization of LanA and in epithelial apico-basal polarity. The defects resemble those observed in follicle cells (that is, epithelial cells covering the fly oocyte) mutant for crag, a gene encoding for a protein localized in early and recycling endosomes and proposed to regulate protein transport and membrane deposition of BM proteins. Zygotic removal of crag induced anomalous localization of LanA on the apical side of AS cells. Zygotic or both maternal and zygotic removal of crag produced cuticles with a weak dorsally wrinkled phenotype, suggesting that Crag has a redundant function with another protein in the fly embryo and that the low BM levels of LanA observed in these embryos are sufficient to exert their function. Interestingly, halving the dose of scarf expression gave rise to a large proportion of crag cuticles with phenotypes similar to those caused by scarf depletion (Sorrosal, 2010).

Antibodies against Scarf were raised to analyse its subcellular localization. As the antibodies did not work properly at embryonic stages and did not detect endogenous levels of Scarf protein, the wing imaginal disc, a monolayered epithelium, was used to analyse its subcellular localization. Scarf was not detected in wild-type wing cells. When scarf and green fluorescent protein (GFP) were expressed in a restricted domain in the wing disc, Scarf was detected not only in the GFP-labelled scarf-producing cells, but also on the apical side of the epithelium at long distances from the source. When atrial natriuretic factor-GFP (a fusion between the secreted rat atrial natriuretic peptide and GFP) was expressed, the protein was also observed at long distances from the source; however, it did not accumulate on the apical side of the epithelium. Scarf was found to localize in small punctate structures throughout the cytoplasm of scarf non-producing cells. These structures corresponded either to Rab5-positive early endosomes, to Rab7-positive late endosomes, or to Rab11-positive recycling endosomes. Together, these results indicate that Scarf is secreted apically and is internalized through endocytosis by non-producing cells. To confirm that Scarf is a secreted protein, Drosophila S2 cells were transfected with either a scarf-myc-tagged transgene or a transgene driving the expression of a myc-tagged membrane-tethered form of Scarf (Scarf-CD2). Scarf was isolated not only from the protein extract of the scarf-myc-expressing cells but also from the supernatant. Transfected Scarf-CD2 and endogenous actin were not observed in the supernatant (Sorrosal, 2010).

This study has characterized the role of scarf, a JNK-regulated gene, in GBR and DC, two morphogenetic processes that rely on cell-matrix interactions between the AS epithelium and neighbouring cell populations. Evidence that Scarf is a secreted protein expressed in LE cells and involved in promoting the localized accumulation of LanA in the BM of AS cells. Although low levels of Scarf were detected in AS cells, the cuticle phenotype of scarf mutant embryos and the defects observed in BM localization of LanA and epithelial integrity of AS cells were rescued largely by driving expression of a scarf transgene only in ectodermal cells, indicating that Scarf is acting as a secreted protein. Three alternative hypotheses might explain the role of Scarf and Crag in the polarized localization of LanA to the BM. As Crag and Scarf are localized specifically on the apical side of the epithelium, they would have a repulsive role, inhibiting the targeting of LanA-containing vesicles with apical membranes. Alternatively, LanA might be secreted apically and basally and be stabilized preferentially on the basal side or degraded on the apical side. As Scarf encodes for a putative serine protease-like protein without catalytic activity, Scarf would function, in this scenario, to facilitate the effect of a cascade of serine proteases involved in the degradation of LanA in the apical domain of epithelial cells. Finally, in scarf mutant cells, LanA might be transported from the basal to the apical side through transcytosis, a mechanism responsible for the formation of a small apical BM cap over pre-invasive epithelial cells during Drosophila oogenesis (Sorrosal, 2010).

Another secreted serine protease-like protein without catalytic activity, Masquerade, regulates cell-matrix interactions at the somatic-muscle attachment in the fly embryo, a process that also depends on integrin and laminin activity. It is speculated that a number of non-functional serine protease-like proteins, such as Masquerade and Scarf, are regulated temporally and spatially to promote the appropriate localization of BM proteins in a context-dependent manner to facilitate cell-matrix interactions and to ensure the maintenance of epithelial integrity. Similarly, among the large class of vertebrate functional and non-functional serine protease-like proteins involved in remodelling the extracellular matrix, some of them might exert a similar action (Sorrosal, 2010).

Apical deficiency triggers JNK-dependent apoptosis in the embryonic epidermis of Drosophila

Epithelial homeostasis and the avoidance of diseases such as cancer require the elimination of defective cells by apoptosis. This study investigated how loss of apical determinants triggers apoptosis in the embryonic epidermis of Drosophila. Transcriptional profiling and in situ hybridisation show that JNK signalling is upregulated in mutants lacking Crumbs or other apical determinants. This leads to transcriptional activation of the pro-apoptotic gene reaper and to apoptosis. Suppression of JNK signalling by overexpression of Puckered, a feedback inhibitor of the pathway, prevents reaper upregulation and apoptosis. Moreover, removal of endogenous Puckered leads to ectopic reaper expression. Importantly, disruption of the basolateral domain in the embryonic epidermis does not trigger JNK signalling or apoptosis. It is suggested that apical, not basolateral, integrity could be intrinsically required for the survival of epithelial cells. In apically deficient embryos, JNK signalling is activated throughout the epidermis. Yet, in the dorsal region, reaper expression is not activated and cells survive. One characteristic of these surviving cells is that they retain discernible adherens junctions despite the apical deficit. It is suggested that junctional integrity could restrain the pro-apoptotic influence of JNK signalling (Kolahgar, 2011).

This study has shown that apical, but not basolateral, disruption leads to apoptosis in a developing epithelium. It was also showed that JNK signalling is a key intermediate in the signal transduction mechanism that triggers apoptosis in response to the loss of apical determinants. Apical disruption leads to activation of JNK signalling, which in turn activates transcription of the pro-apoptotic gene rpr. Moreover, rpr expression is not activated in apically disrupted embryos that are prevented from activating JNK signalling. Interestingly, in bicoid-deficient embryos and other segmentation mutants, cell fate misspecification requires activation of a different pro-apoptotic gene, hid. Therefore, distinct quality control pathways might exist to ensure that different forms of defective cells are removed from developing epithelia. JNK signalling has been shown to mediate apoptosis in a variety of other situations, including after DNA damage. However, JNK signalling does not necessarily cause apoptosis. Indeed, this pathway modulates many other cell activities, such as proliferation, differentiation and morphogenesis. What conditions determine whether JNK triggers apoptosis or not is an important issue. Another obvious question raised by the current findings concerns the nature of the mechanism that triggers JNK signalling following the loss of apical determinants (Kolahgar, 2011).

This study has shown that, in the embryonic epidermis, JNK signalling is activated by the loss of apical, not basolateral, determinants. In fact, reduction of lgl activity prevents JNK activation in crb mutant embryos. Similarly, Scrib knockdown prevents JNK activity in the mouse mammary epithelium, suggesting that the loss of the basolateral domain could have a general anti-JNK (and perhaps anti-apoptotic) activity. Although JNK activation has been documented in tissues that lack a basolateral determinant, it is suggested that this might be an indirect consequence of cell competition, which triggers JNK signalling, or of the specific experimental conditions (partial reduction of Puc activity). Overall, the results suggest the existence of an apical domain-dependent activity that modulates JNK signalling in the embryonic epidermis of Drosophila. This activity could be similar to that postulated to be at work in cultured MDCK cells, but is likely to be distinct from the process that leads to apoptosis in response to mosaic disruption of the basolateral domain in imaginal discs (Kolahgar, 2011).

The mechanism underlying the activation of JNK signalling by loss of apical determinants remains unknown. For example, it is uncertain at this point whether there is an apically localised activity that directly modulates JNK signalling or whether a more indirect route is at work (paths 1 and 2 respectively; see Signalling upstream and downstream of JNK). Since apical organisation is required for the establishment of adherens junctions, it is conceivable that the effect of apical disruption on JNK signalling is mediated by junctional disruption. This possibility is compatible with the absence of ectopic JNK activation in basolateral mutants, in which E-cadherin remains localised to patches at the cell surfac. However, one would have to invoke that slight junctional disruption is sufficient to trigger JNK signalling, as this pathway is upregulated in the dorsal region of crb mutants, where the extent of junctional disruption is relatively mild. It has not been possible to discriminate between paths 1 and 2, partly because of the current difficulty in eliminating adherens junctions from early Drosophila embryos. Future work will require novel means of interfering specifically with adherens junctions. Considering the lack of involvement of Egr, it will also be necessary to identify the upstream components of JNK signalling that respond to epithelial disruption (Kolahgar, 2011).

Overexpression of Puc, a feedback inhibitor of JNK signalling, prevents apoptosis in the ventral epidermis of crb embryos. This is clear evidence that JNK signalling is required for apical deficit to trigger apoptosis. However, it is well established that JNK signalling does not necessarily lead to apoptosis. This is particularly well illustrated by the situation at the dorsal edge of wild-type embryos, where JNK is highly active without triggering apoptosis. Moreover, in crb mutants, a 6- to 10-cell-wide band of dorsal tissue is refractory to the pro-apoptotic influence of JNK signalling. Therefore, additional conditions must be met for JNK signalling to activate rpr expression and trigger apoptosis. This study has identified two situations when refractory cells succumb to the pressure of JNK signalling (see Signalling upstream and downstream of JNK). One involves the reduction of Puc and the other the removal of zygotic E-cadherin activity. The first situation suggests that endogenous Puc can limit the ability of JNK to activate rpr expression and trigger apoptosis. The important role of Puc in preventing cell death is also highlighted by the extensive apoptosis seen in embryos lacking both maternal and zygotic Puc activity. Puc could act solely by limiting the extent of JNK signalling, thus preventing the very high level of signalling required for rpr expression. Alternatively, or in addition, Puc could have an activity that specifically prevents certain genes, such as rpr, from being spuriously activated. In any case, it is likely that the regulatory relationships between JNK, Puc and apoptosis are influenced by the cellular context (e.g., the state of adherens junctions (Kolahgar, 2011).

JNK signalling triggers rpr expression (and apoptosis) more readily if adherens junctions are weakened or disrupted. Therefore, junctional integrity could also protect epithelial cells from the pro-apoptotic effects of JNK signalling. Dorsoventral differences in junctional integrity and remodelling have been noted in the embryonic epidermis of Drosophila and these might explain why these two regions respond differently to the loss of crb. The results suggest that residual junctional integrity in the dorsal epidermis prevents JNK signalling from activating rpr expression. It is conceivable that a protective signal emanates from adherens junctions. Alternatively, junctional disruption could interfere with the ability of Puc to rein in the effect of JNK signalling on rpr expression. Although differential junctional dynamics between the dorsal and ventral epidermis could determine the propensity to undergo apoptosis, the possibility cannot be excluded that other dorsoventral determinants are at work too (Kolahgar, 2011).

This study has shown that loss of apical polarity leads to apoptosis by activating JNK signalling and causing junctional disruption. It is expected that this response, which is readily detectable in the crb mutant condition, might reflect a process that ensures the removal of abnormal and damaged cells during epithelial homeostasis. It is hoped that understanding the machinery that links epithelial disruption to JNK signalling and the transcription of pro-apoptotic genes will suggest means of reactivating this pathway in pathological situations (Kolahgar, 2011).

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date revised: 5 October 2012

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