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Transcriptional Regulation

Beta-galactosidase expression by the pucE69 insertion is abolished in hemipterous and basket mutants (Glise, 1995; Riesgo-Escovar, 1996). Beta-galactosidase activity is enhanced after overexpression of activated forms of Drac1 (DRac1V12) and Dcdc42 (Dcdc42V12) (Glise, 1997) that activate JNK signaling. As puc encodes a JNK phosphatase that appears to down-regulate the JNK pathway, the expression of puc (beta-galactosidase) was studied in puc mutants, in which JNK activity is enhanced. The misregulation of pucE69 LacZ activity has been reported previously (Ring, 1993 and Glise, 1997). All puc insertional alleles in mutant embryos show higher levels and more cells expressing beta-galactosidase than wild type, as for example, in the leading edge of the epidermis, the amnioserosa, the ectoderm, and the nervous system (Martin-Blanco, 1998).

The requirement for the DJNK pathway to activate puc expression is also mirrored in the reduction of JNK phosphatase activity in mutants for hep: JNK activity is reduced threefold in hep1 mutants extracts, as expected for a loss of function in a JNK activator. In addition, extracts from hep1 mutants show less phosphatase activity on exogenous JNK (a 50% reduction), but identical levels of ERK phosphatase function, compared with wild-type embryo extracts (Martin-Blanco, 1998).

Altogether, these results support a role for the Drosophila JNK pathway in the control of puc expression. puckered expression itself is a consequence of the activity of the JNK pathway. During dorsal closure, JNK signaling has a dual role: to activate an effector, encoded by decapentaplegic, and as an element of negative feedback regulation encoded by puckered (Martin-Blanco, 1998).

The small GTPases Rac and Rho act as cellular switches in many important biological processes. In the fruit fly Drosophila, RhoA participates in the establishment of planar polarity, a process mediated by the receptor Frizzled (Fz). Thus far, analysis of Rac in this process has not been possible because of the absence of mutant Rac alleles. The roles of Rac and Rho in establishing the polarity of ommatidia in the Drosophila eye were investigated. By expressing a dominant negative or a constitutively activated form of Rac1, Rac signaling was interfered with specifically and ommatidial polarity was disrupted. The resulting defects are similar to the loss/gain-of-function phenotypes typical of tissue-polarity genes. Through genetic interaction and rescue experiments involving a polarity-specific, loss-of-function dishevelled (dsh) allele, Rac1 was found to act downstream of Dsh in the Fz signaling pathway, but upstream of, or in parallel to, RhoA. Rac signals to the nucleus through the Jun N-terminal kinase (JNK) cascade in this process. By generating point mutations in the effector loop of RhoA, it was found that RhoA also signals to the nucleus during the establishment of ommatidial polarity. Nevertheless, Rac and RhoA activate transcription of distinct target genes. Thus Rac is specifically required downstream of Dsh in the Fz pathway. It functions upstream or in parallel to RhoA and both signal to the nucleus, through distinct effectors, to establish planar polarity in the Drosophila eye (Fanto, 2000).

To better characterize nuclear signaling by Rac and RhoA, the expression of puckered (puc) and Delta (Dl) were studied. Dl is the only known transcriptional target of Fz signaling in R3, and puc-lacZ expression serves as a measure of JNK activity in vivo. The puc gene is a transcriptional target of JNK signaling in Drosophila, and encodes a dual specificity protein phosphatase that acts as a negative regulator of JNK itself in a feedback loop. In the wild type, very weak beta-galactosidase expression from the puc enhancer trap line is detectable in all photoreceptor precursors. Expression of sev;racV12 lead to strong upregulation of puc-lacz in one or, more frequently, two cells of the cluster, identified as R3/R4 precursor cells, consistent with the expression pattern of sev;RacV12. These data resemble the upregulation of puc-lacz when the JNK pathway has been activated in the same cells (Fanto, 2000).

In contrast, RhoV14 affects puc-lacz expression differently. Although in sev;RhoV14 eye discs puc-lacz expression is upregulated in some cells at a later stage, these were not identifiable as the R3/R4 pair, but were often found in the position of the R2/R5/R8 precursors (where sev is not expressed). This suggests that the effect seen is not a direct consequence of Rho activation, but more likely a secondary effect (RhoAV14 E40L fails to induce significant puc-lacz expression). Thus, the direct transcriptional activation of puc-lacz in R3/R4 correlates with the genetic interactions with the JNK module, suggesting a difference in the action of Rac and RhoA (Fanto, 2000).

An important aspect of R3/R4 cell fate and ommatidial polarity determination is the upregulation of Dl expression in the R3 precursor by Fz. Dl then signals to Notch on the R4 precursor, resulting in the choice of the R4 cell fate. In addition to Fz, other components of the Fz/planar-polarity pathway have also been found to upregulate Dl transcription. Thus, whether Rac and RhoA also regulate Dl transcription was investigated by monitoring Dl-lacZ expression in sev;RacV12 and sev;RhoV14 eye discs (Fanto, 2000).

In the wild type, Dl is expressed dynamically in photoreceptor precursors behind the furrow. Within the R3/R4 pair, it is expressed in R3 from rows 4 to 8, whereas it remains at lower levels in R4. In contrast to the difference in puc expression, both sev;RacV12 and sev;RhoV14 upregulated Dl-lacz expression in both R3/R4 precursors. The RhoAV14 E40L isoform that is impaired in nuclear signaling does not affect Dl expression, confirming the importance of nuclear signaling by RhoA. These effects are very similar to those of sev;Fz, supporting the idea that Rac and RhoA act downstream of Fz in the regulation of the R3/R4 cell fate. Their different effects on puc-lacz indicate that their downstream effectors in nuclear signaling are distinct (Fanto, 2000).

Little is known about the exact role that hemipterous and basket/DJNK play in the process of dorsal closure. Specifically, it is not clear what the target of JNK phosphorylation is in dorsal closure. JNK could directly phosphorylate and modify cytoskeletal components involved in dorsal closure such as Zipper (Nonmuscle myosin), Coracle (a Drosophila homolog of the vertebrate band 4.1 cytoskeletal protein), Inflated or Myospheroid (integrins involved in cell adhesion). Alternatively JNK could modify the activity of transcription factors that are known to be involved in dorsal closure (see Jun-related antigen and Anterior open/Yan). Mutations in genes coding for several transcripiton factors have dorsal open phenotypes, like pannier and serpent (two GATA transcript factors), and anterior open (an ETS domain protein). The fact that both hep and bsk mutants affect the expression of puckered (a gene with a dorsal closure phenotype), suggests that JNK and HEP act by regulating transcription factors rather than by directly modifying cytoskeletal components involved in the actual process of cell shape change (Rieso-Escovar, 1996).

Drosophila kayak mutant embryos exhibit defects in dorsal closure, a morphogenetic cell sheet movement during embryogenesis. It is shown that kayak encodes D-Fos, the Drosophila homologue of the mammalian proto-oncogene product, c-Fos. D-Fos is shown to act in a similar manner to Drosophila Jun: in the cells of the leading edge it is required for the expression of the TGFbeta-like Decapentaplegic (Dpp) protein, which is believed to control the cell shape changes that take place during dorsal closure. The kayak expression domain include the cells of the amnioserosa and the lateral epidermis during the process of dorsal closure. At the onset of dorsal closure, elevated levels of D-Fos can be detected in the nuclei of leading-edge cells as they initiate elongation. Defects observed in mutant embryos, and adults with reduced Fos expression, are reminiscent of phenotypes caused by 'loss of function' mutations in the Drosophila JNKK homologue, hemipterous. Another downstream effect of Bsk signaling, the transcriptional activation of the puckered gene (puc) in the cells of the leading edge is also abrogated in kay1 mutant embryos. Taken together, the requirement of both D-Fos and D-Jun for dpp and puc expression in leading-edge cells suggests that the JNK signal is relayed by a heterodimeric transcription factor composed of D-Jun and D-Fos. These results indicate that D-Fos is required downstream of the Drosophila JNK signal transduction pathway, consistent with a role in heterodimerization with D-Jun, to activate downstream targets such as dpp (Zeitlinger, 1998).

In Drosophila, the Jun-N-terminal Kinase-(JNK) signaling pathway is required for epithelial cell shape changes during dorsal closure of the embryo. In the absence of JNK pathway activity, as in the DJNKK/hemipterous (hep) mutant, the dorsolateral ectodermal cells fail both to elongate and move toward the dorsal midline, leading to dorsally open embryos. hep and the JNK pathway are required later in development, for correct morphogenesis of other epithelia, the imaginal discs. During metamorphosis, the imaginal discs undergo profound morphological changes, giving rise to the adult head and thoracic structures, including the cuticle and appendages. hep mutant pupae and pharate adults show severe defects in disc morphogenesis, especially in the fusion of the two lateral wing discs. These defects are accompanied by a loss of expression of puckered (puc), a JNK phosphatase-encoding gene, in a subset of peripodial cells that ultimately delineates the margins of fusing discs. In further support of a role for puc in disc morphogenesis, pupal and adult hep phenotypes are suppressed by reducing puc function, indicative of a negative role for puc in disc morphogenesis. Furthermore, the small GTPase Dcdc42, but not Drac1, is an activator of puc expression in a hep-dependent manner in imaginal discs. Altogether, these results demonstrate a new role for the JNK pathway in epithelial morphogenesis, and provide genetic evidence of a role for the peripodial membrane in disc morphogenesis. A general model is discussed whereby the JNK pathway regulates morphogenesis of epithelia with differentiated edges (Agnès, 1999).

The puckered (puc) gene encodes a JNK MAPK phosphatase that negatively regulates the activity of the JNK pathway during dorsal closure. It is specifically expressed in a population of lateral cells (the leading edge) that delineates the boundary between the ectoderm and the amnioserosa. Importantly, puc expression is activated by hep in the leading edge, to initiate a negative feedback loop. Therefore, puc is a marker of both JNK activity and the margins of moving epithelia during dorsal closure. It was therefore of particular interest to analyze the expression pattern of puc during larval and pupal development, using the pucE69 allele, a P lacZ enhancer-trap line inserted in the puc gene. In the wild type, expression of puc becomes detectable in the third instar larva (epidermis and spiracles) and slightly increases throughout this stage. It then becomes stronger during prepupariation and decreases by the end of this stage. Interestingly, puc is specifically expressed in particular cell populations in all thoracic discs. In the proximal part of the wing, haltere and leg discs, puc is strongly expressed in the stalk region, where imaginal discs connect to the larval epidermis. Further, puc is also expressed in rows of cells in wing, eye-antenna, haltere and leg discs, in a pattern that is reminiscent of the margin expression observed in the ectoderm during dorsal closure. These cells are on the dorsal side of imaginal discs, and are part of a particular structure of the discs, the peripodial epithelium or peripodial membrane. The peripodial membrane is an epithelium made of squamous cells easily distinguishable from those of the disc proper due to their larger and more widely spaced nuclei. During metamorphosis, cells of the peripodial membrane play an active role through the dramatic change of their shape, either by intensive stretching or contraction. In addition to this role, the peripodial membrane also contributes to some parts of the adult integument, especially in regions where adjacent discs will suture. To confirm that puc is expressed in the peripodial epithelium, double immunostaining was performed using an antibody directed against beta-galactosidase (reflecting puc expression) and another against the conserved N terminus of the Broad-complex (Br-C) protein isoforms. Br-C is ubiquitously expressed in the nuclei of disc cells, and serves here as a marker of peripodial membrane vs columnar epithelial cells. The results show that puc is expressed in the peripodial membrane of all thoracic discs, at the boundary between peripodial and columnar epithelia. Due to a more sensitive detection using antibody staining, it was found that puc is expressed in a larger subset of peripodial membrane cells than that revealed using X-gal stainings. These experiments thus confirm the restriction of puc expression in the peripodial membrane. Later during prepupariation, puc expression is maintained in the peripodial membrane and marks the presumptive suture sites of imaginal discs with their neighbors. By the end of this stage, puc staining is found at the frontier between sutured discs. These data suggest that puc, and the JNK pathway, are required in a specific subset of peripodial cells for morphogenesis of imaginal discs (Agnès, 1999).

In embryos, hep activates puc and dpp expression in ectodermal margins. puc activation serves to initiate a negative feedback loop that controls the levels of JNK activity during dorsal closure. To test whether these important regulatory links also exist during disc morphogenesis in larvae, the expression pattern of puc and dpp in L3 larvae were analyzed using strong hep alleles. Both in hepr39 and hepr75male larvae, puc expression in imaginal tissues is dramatically reduced. Since the loss of puc staining may reflect a delay in the onset of puc expression, white-pupae were dissected and stained to reveal puc-lacZ expression. As in L3 larval stage, puc expression is absent in hep white-pupa discs, whereas wild-type puc expression slightly increases at this developmental stage. In contrast, puc expression is still detectable in the epidermis, mouthparts, and spiracles both in the wild type and in hep mutants, indicating a hep-independent expression of puc in these tissues as well as providing an internal control. In contrast, a dpp-lacZ line, which is expressed in a pattern very similar to that of puc in the peripodial membrane, is not controlled by hep function in L3 and 5 h APF discs. Similarly, in later stage discs (L3 to 8 hours APF), dpp expression is not changed in regions close to the dorsal midline, like the notum. Together, these results show that hep is required for the normal expression of puc in the peripodial membrane of thoracic discs, suggesting that the subset of puc-expressing cells in the peripodial membrane is the primary site of JNK activity during disc morphogenesis. They also highlight an important difference with the situation found during dorsal closure, where the JNK and dpp pathways are coupled (Agnès, 1999).

During the process of dorsal closure, hep and puc have opposing effects on their common target, basket/DJNK. To begin to analyse the role of puc during disc morphogenesis, genetic interactions between hep and puc were examined. In addition to dominantly suppressing the embryonic lethality associated with the hep1allele, puc mutations also strongly suppress the adult phenotypes associated with this allele. The extent of suppression allows a normally embryonic lethal stock (hep1/hep1) to be kept. Moreover, reduction by half of puc activity also suppresses hepr early pupal development arrest, allowing a large proportion of hemizygous males to proceed to late pupal stage. In the presence of only one copy of puc, defects associated with mutant hep are strongly reduced. These results indicate that puc has a negative regulatory function in discs, suggesting that the regulatory link established between hep and puc during dorsal closure is well conserved in imaginal discs during metamorphosis (Agnès, 1999).

The small GTPases of the Rho family, DRac1 and Dcdc42, can positively regulate the Drosophila JNK pathway in embryos. To further investigate the conservation of JNK pathway function in disc morphogenesis, activated forms of DRac1 (UAS-Drac1V12) and DCdc42 (UAS-DCdc42V12) were expressed in different subsets of imaginal disc cells using the UAS-GAL4 system. Because GAL4 lines driving specific expression in the peripodial membrane do not exist, GAL4 lines that are expressed in different domains of the columnar epithelium were used to test for a potential activating role of Drac1 and Dcdc42 in discs; among those, some are also expressed in the peripodial epithelium. Targetted expression of either Drac1 or Dcdc42 results in a strong ectopic expression of puc in patterns specific of each GAL4 driver used. In the wing, expression is prominent along the anteroposterior boundary. The effects of Drac1 and Dcdc42 on puc are similar, although not exactly the same in terms of the activation levels and pattern of activated cells. In a hepr mutant background, only Dcdc42-mediated puc ectopic expression is strongly suppressed, indicating that hep is required downstream of Dcdc42 to activate puc. However, suppression is not complete, since puc expression remains in some cells of the discs, indicating a hep-independent activation of puc. Similar results were also observed in embryos. In addition to its potent activity on puc expression, Dcdc42 also induces dramatic defects in the overall disc morphology, including a reduction of the size and an aberrant shape. These defects are also strongly suppressed by hep, reinforcing the notion that a Dcdc42, hep, puc cascade plays an important morphogenetic activity in imaginal discs. These data suggest that Dcdc42 may play a role in disc morphogenesis in combination with hep and puc. In addition, they provide evidence that cells of the columnar epithelium are competent for JNK activity, and that a limiting factor for JNK activation lies upstream of the small GTPases. In this respect, the disc epithelium is very similar to the lateral ectodermal cells during dorsal closure (Agnès, 1999).

The fact that hep regulates puc expression in cells of the peripodial membrane indicates a requirement of the JNK pathway in these cells, and reveals at least one site of JNK activity during metamorphosis. The role of this structure, which covers one side of the larval imaginal discs, has been controversial and unclear. It has been proposed to play an active role during metamorphosis, by undergoing successive stretching and contraction. The results provide a genetic confirmation of a role for the peripodial membrane, by showing a clear link between abnormal disc morphogenesis and spreading, and cell fate determination in the peripodial membrane mediated by the JNK pathway (Agnès, 1999).

One important question is whether the defects observed in hep mutants are only the result of a lack of peripodial membrane function. This question could be addressed directly by generating mutant hep clones in the peripodial membrane specifically, using directed mosaics. If hep function is indeed restricted to the peripodial membrane, then it would strongly argue in favor of a model in which the peripodial membrane would orchestrate the process. Such a role has been proposed in dorsal closure of the leading edge. Interestingly, a Hedgehog-mediated organizing role of the peripodial membrane during regeneration of the T2 leg disc has recently been reported, as well as a novel structural basis for the long-range activity of signaling molecules in the wing disc. Whether hep and the JNK pathway contribute to metamorphosis through related mechanisms will be further investigated (Agnès, 1999).

In Drosophila, the Jun amino-terminal kinase (JNK) homolog Basket (Bsk) is required for epidermal closure. Mutants for Src42A, a Drosophila c-src protooncogene homolog, are described. Src42A functions in epidermal closure during both embryogenesis and metamorphosis. The severity of the epidermal closure defect in the Src42A mutant depends on the amount of Bsk activity, and the amount of Bsk activity depends on the amount of Src42A. Thus, activation of the Bsk pathway is required downstream of Src42A in epidermal closure. This work confirms mammalian studies that demonstrate a physiological link between Src and JNK (Tateno, 2000).

Genes that regulate cell shape changes in Drosophila are required for dorsal closure of the embryonic epidermis and thorax closure of the pupal epidermis. Mutations in genes such as hemipterous (hep) and basket (bsk, also known as DJNK) result in abnormal embryos with a dorsal hole or abnormal adults with a dorsal midline cleft. Hep and Bsk are homologous to the mammalian MKK7 (MAPK kinase 7) and JNK, and they are components of a MAPK (mitogen-activated protein kinase) cascade. Although the role of the Hep-Bsk cascade during dorsal closure has been extensively studied, the upstream trigger of this cascade is poorly understood. To identify the trigger, a screen was carried out for mutants showing the dorsal midline cleft phenotype, like a mild hep mutant. The mutant for Src42A shows this phenotype and Src42A regulates Bsk during Drosophila development (Tateno, 2000).

Furthermore, the Tec29 Src42A double mutant shows complete embryonic lethality, and a certain fraction of the dead embryos show the dorsal open phenotype. Activated DJun, a transcription factor downstream of Bsk, partially rescues the dorsal open phenotype in the Tec29 Src42A double mutant. Thus, Src42A appears to regulate Bsk in the fusion of epithelial sheets during embryogenesis and metamorphosis, and Tec29 is involved in this regulation. The double mutant for Src64 and Src42A manifests a mild but clear dorsal open phenotype, which suggests a functional redundancy between Src64 and Src42A (Tateno, 2000).

Expression of puc is known to be induced by the Bsk signal. In the wing disc of the wild-type third-instar larva, puc is expressed in the dorsal midline of the adult notum. In the wing disc of the Src42AJp45 mutant, puc expression is reduced. In contrast, larvae with a constitutively activated form of Src42A (Src42ACA) shows ectopic expression of puc. Further, introduction of a hep null mutation reduces the amount of ectopic puc expression. It is known that Bsk induces expression of puc and decapentaplegic (dpp) during embryonic dorsal closure. The embryos of the Tec29 Src42A double mutant do not show any puc or dpp expression in the leading edge cells. These results indicate that Src42A, Tec29, Hep, and Bsk regulate dpp and puc expression during embryonic dorsal closure (Tateno, 2000).

During embryonic dorsal closure, the Hep-Bsk signal is required for elongation of the leading edge cells. In the absence of the Bsk signal, these cells do not fully elongate. The accumulation of F-actin and phosphotyrosine (P-Tyr) in leading edge cells is associated with the elongation of these cells. Accumulation of these substances is disturbed in the DJun and the puc mutants. In the double mutant for Tec29 and Src42A, the leading edge cells contain reduced quantities of F-actin and P-Tyr, and these cells are only partially elongated. Thus, the defect in embryonic dorsal closure in the Tec29 Src42A double mutant is caused by this failure in cell shape change, as is the case in the DJun mutant (Tateno, 2000).

A model is proposed in which Src42A, upon receiving an unidentified signal, activates the Hep-Bsk pathway to regulate cell shape change and epidermal layer movement. This is consistent with the observation in mammals that c-Src regulates the cell morphogenetic and migratory processes and is known to activate JNK. As in Drosophila, c-Src definitely affects F-actin organization and P-Tyr localization during cell morphogenesis. Therefore, Src regulation of JNK activity toward a change in cell shape may be conserved (Tateno, 2000).

It can be also interpreted that Src42A acts upstream of DFos, a dimerization partner of DJun. Although the Src42A, Tec29, and Src64 single mutants do not show a dorsal open phenotype, the DFos mutant clearly exhibits it. This relationship is also analogous to that in mammals. Both c-src and c-fos knockout mice have a similar defect, osteopetrosis caused by reduced osteoclast function. But the phenotypic severity is milder in c-src than in c-fos knockouts; this can be explained by the functional overlap in multiple Src-family tyrosine kinases. Accordingly, in both Drosophila and mammals, multiple nonreceptor tyrosine kinases may cooperate to regulate the function of the Jun/Fos complex (Tateno, 2000).

The TAK kinases belong to the MAPKKK group and have been implicated in a variety of signaling events. Originally described as a TGFß activated kinase (TAK), the mammalian protein has, however, been demonstrated to signal through p38, Jun N-terminal kinase (JNK) and Nemo types of MAP kinases, and the NFkappaB inducing kinase. Despite these multiple proposed functions, the in vivo role of TAK family kinases remains unclear. The isolation and genetic characterization of the Drosophila TAK homolog (TGF-ß activated kinase 1: Tak1) is reported in this study. Sequence analysis reveals a 678 amino acid long open reading frame (ORF), which shows the highest similarity to vertebrate TAK proteins. Subsequent conceptual translation displays an N-terminal kinase domain of about 280 amino acids, showing 54% identity and 69% similarity to mTAK1, and a long C-terminal domain. The C-terminal domain is less well conserved. However, a 60 amino acid stretch shows a significant level of conservation as compared to the vertebrate and C. elegans orthologs (36% identity and 60% similarity to mTAK1), constituting a conserved protein-protein interaction interface for putative modulators of TAK activity, such as TAB-2 (Mihaly, 2001).

The use of overexpression and double-stranded RNA interference (RNAi) techniques has allowed analysis of Tak1 function during embryogenesis and larval development. Overexpression of Tak1 in the embryonic epidermis is sufficient to induce the transcription of the JNK target genes decapentaplegic and puckered. Furthermore, overexpression of dominant negative (DN) or wild-type forms of Tak1 in wing and eye imaginal discs, respectively, results in defects in thorax closure and ommatidial planar polarity, two well described phenotypes associated with JNK signaling activity. Surprisingly, RNAi and DN-Tak1 expression studies in the embryo argue for a differential requirement of Tak1 during developmental processes controlled by JNK signaling, and a redundant or minor role of Tak1 in dorsal closure. In addition, Tak1-mediated activation of JNK in the Drosophila eye imaginal disc leads to an eye ablation phenotype due to ectopically induced apoptotic cell death. Genetic analyses in the eye indicate that Tak1 can also act through the p38 and Nemo kinases in imaginal discs. These results suggest that dTAK can act as a JNKKK upstream of JNK in multiple contexts and also other MAPKs in the eye. However, the loss-of-function RNAi studies indicate that it is not strictly required and thus either redundant or playing only a minor role in the context of embryonic dorsal closure (Mihaly, 2001).

Dorsal closure, taking place in mid-embryogenesis, describes the morphogenetic movements of the epidermis in order to replace the amnioserosa on the dorsal side of the embryo. This event is driven by the concerted spreading of epidermal cells towards the dorsal midline, where the two contralateral epidermal cell layers meet and remain connected. The JNK signaling module and nuclear targets of JNK, the AP-1 transcription factors dJun and dFos, control the process of dorsal closure. Uncompleted or failed dorsal closure is indicative of disrupted JNK signaling. Mutations in all known components of the JNK signaling pathway result in dorsal open embryos. During dorsal closure, the expression of the dpp and puc genes in cells of the leading edge is controlled by the JNK kinase module and the AP-1 transcription factors Jun and Fos. Leading edge cells show loss of puc and dpp expression when deficient for JNK signaling. Conversely, constitutive activation of JNK signaling in the embryonic epidermis by overexpressing activated Rac or Cdc42 induces the upregulation of dpp and puc. To address the question of whether dTAK can activate and act through the JNK MAPK module, Tak1 was expressed under the control of the en-GAL4 and pnr-GAL4 drivers and the induction of dpp and puc was monitered in the epidermis of stage 12-15 embryos (pnr is strongly expressed in leading edge cells and cells neighboring the leading edge). In wild-type, dpp is expressed in two lateral stripes along the Drosophila embryo, and the dorsal most stripe corresponds to the leading edge of the epidermis. Overexpression of Tak1 with either GAL4 driver causes ectopic upregulation of dpp, as monitored by RNA in situ hybridization. Similarly, the analysis of embryos carrying one copy of a puc lacZ enhancer trap by ß-galactosidase activity staining shows a clear and robust ectopic puc expression when Tak1 is overexpressed. These patterns of dpp and puc activation by Tak1 are identical to those observed with activated Jun, suggesting that the effect is direct and mediated by the JNK signaling pathway (Mihaly, 2001). In summary, these data indicate that overexpression of Tak1 in the embryonic ectoderm is sufficient to induce high-level expression of both known JNK target genes. Since the same upregulation of puc and dpp is observed with activated JNKK/Hep and Jun (a JNK activated transcription factor), they strongly suggest that Tak1 acts through the JNK/ Jun(AP-1) module in the context of dorsal closure (Mihaly, 2001).

During Drosophila oogenesis, the formation of the egg respiratory appendages and the micropyle require the shaping of anterior and dorsal follicle cells. Prior to their morphogenesis, cells of the presumptive appendages are determined by integrating dorsal-ventral and anterior-posterior positional information provided by the epidermal growth factor receptor (EGFR) and Decapentaplegic (Dpp) pathways, respectively. Another signaling pathway, the Drosophila Jun-N-terminal kinase (JNK) cascade, is essential for the correct morphogenesis of the dorsal appendages and the micropyle during oogenesis. Mutant follicle cell clones of members of the JNK pathway, including DJNKK/hemipterous (hep), DJNK/basket (bsk), and Djun, block dorsal appendage (DA) formation and affect the micropyle shape and size, suggesting a late requirement for the JNK pathway in anterior chorion morphogenesis. In support of this view, hep does not affect early follicle cell patterning as indicated by the normal expression of kekkon (kek) and Broad-Complex (BR-C), two of the targets of the EGFR pathway in dorsal follicle cells. Furthermore, the expression of the TGF-ß homolog dpp, which is under the control of hep in embryos, is not coupled to JNK activity during oogenesis. hep controls the expression of puckered (puc) in the follicular epithelium in a cell-autonomous manner. Since puc overexpression in the egg follicular epithelium mimics JNK appendages and micropyle phenotypes, it indicates a negative role of puc in their morphogenesis (Suzanne, 2001).

Efficient wound healing including clotting and subsequent reepithelization is essential for animals ranging from insects to mammals to recover from epithelial injury. It is likely that genes involved in wound healing are conserved through the phylogeny and therefore, Drosophila may be a useful in vivo model system to identify genes necessary during this process. Furthermore, epithelial movement during specific developmental processes, such as dorsal closure (DC), resembles that seen in mammalian wound healing. Since puckered (puc) gene is a target of the JUN N-terminal kinase signaling pathway during DC, puc gene expression was investigated during wound healing in Drosophila. puc expression is induced at the edge of the wound in epithelial cells and Jun kinase is phosphorylated in wounded epidermal tissues, suggesting that the JUN N-terminal kinase signaling pathway is activated by a signal produced by an epidermal wound. In the absence of the Drosophila c-Fos homologue, puc gene expression is no longer induced. Finally, impaired epithelial repair in JUN N-terminal kinase deficient flies demonstrates that the JUN N-terminal kinase signaling is required to initiate the cell shape change at the onset of the epithelial wound healing. It is concluded that the embryonic JUN N-terminal kinase gene cassette is induced at the edge of the wound. In addition, Drosophila appears as a good in vivo model to study morphogenetic processes requiring epithelial regeneration, such as wound healing in vertebrates (Ramet, 2002).

In most cases, flies were anesthetized and then mechanically wounded with iridectomy scissors to cut adult abdomen vertically between the third and the seventh tergites. Semi-thin sections were used to examine the histology of wound healing at the cellular level. The first response to epithelial wound is the formation of a clot at the initial site. Subsequently, the clot becomes melanized making the location of the wound clearly visible. The clot appears to consist of an accumulation of melanin and by hemocytes that aggregated at the site of injury. Hemocytes may be involved also in the clearance of cellular debris and invading microbes (Ramet, 2002).

During the first 2 h after wounding no sign of epithelial cell movement can be seen. In most cases, the edges of the cut epidermis are found far away from the broken cuticle. As for the wounded embryonic epidermis, the adult epidermal layer may be submitted to an intrinsic isotropic epidermal tension that retracts it upon any break injury. By 4 h, the epithelial cells of the edge of the wound seem to shed from the disrupted cuticle. These cells appear larger than the epithelial cells lining the normal cuticle, and exhibit cytoplasmic protrusions. By 12 h, the protrusive cytoplasmic extensions extend from the cells of the edge of the wound and 'migrate' toward each other under the melanin clot, Subsequently, they cause the epidermis to form a suture. These cytoplasmic extensions suggest that adult epidermis is healed by the activity of dynamic lamellipodia or filipodia. Correspondingly, cytoskeleton reorganization has been previously described in wound healing model of cultured Madin-Darby canine kidney cell (MDCK) and during Drosophila DC. At this point, the epithelial cells are still enlarged but start to return to their initial shape. The suture of the epithelium is normally achieved within 18 h after injury. By this time, the wounded epithelium has healed, and cells have returned to their original shape (Ramet, 2002).

To ascertain the importance of melanin production in wound healing, the survival rate of Black cells (Bc) homozygous flies was measured after a transversal wound of the adult abdomen cuticle. Bc/Bc flies lack hemolymphatic phenoloxydase activity and therefore, do not produce melanin. By 24 h after wounding, wild type flies and Bc/+ heterozygous flies, present a dominant melanized crystal cell phenotype, but have wild-type phenoloxidase activity, both of which have about 20% mortality, suggesting an efficient wound repair. In contrast, the vast majority (91%) of wounded Bc/Bc mutants died. 50% mortality of Bc flies was already seen by 6 h, suggesting that phenoloxydase activity is essential early in the wound healing process. Similarly, lozenge (lz) mutants, which lack crystal cells and hence present a weak hemolymphatic phenoloxydase activity, exhibit a poor ability to recover from the injury (Ramet, 2002).

The wound clots differently in Bc flies compared to wild type. In wild type flies, a melanin deposit is observed as early as 10 min after wounding and it is still visible 6 h after injury. In contrast, there is no evidence of melanin formation in the wounded integument of Bc flies, indicating that the latter is of hemolymphatic origin. Furthermore, the two edges of the wound are found apart in Bc flies. This failure to keep the edge of the wound in close proximity leads to death due to bleeding. These results underlie the essential role of the phenoloxydase activity, or an associated phenomenon, when it comes to efficient clot formation and the prevention of bleeding (Ramet, 2002).

To determine whether the JNK signaling pathway (which is involved in Drosophila embryonic DC) also participates in wound healing, pucE69, a puc enhancer trap line, was assayed for ß-galactosidase gene expression during wound healing. puc gene expression has been commonly used to monitor the extent of DJNK activation. Cut and uncut epithelium from adult abdomen of wild-type and puc-lacZ flies were separated from the rest of the body, dissected in fixative solution to eliminate gut and fat body, and stained in X-gal solution. Cuticle from injured wild type or control pucE69 flies did not show positive staining. On the contrary, in wounded puc-lacZ flies, the cells close to the wound showed strong induction of puc-lacZ expression, whereas abdominal regions more distant from the wound did not. Semi-thin sections through a puc-lacZ wounded abdomen confirmed epithelial puc expression in wounded tissue. These results suggest that the signaling pathway that activates puc gene expression is induced in epidermal cells close to the wound (Ramet, 2002).

To ascertain that the Drosophila JNK pathway is activated in wounded epidermis, DJun N-terminal kinase activity was assayed using anti-phospho-JNK antibodies. Protein extracts from adult abdominal integument from control and wounded flies were assayed for anti-phospho-JNK reactivity. Phospho-JNK can be detected only in the protein extracts from the wounded epidermis, whereas the unphosphorylated form of DJNK is present in all of the samples. This indicates that DJNK is phosphorylated in response to wounding in epidermal cells, and therefore, suggests that the JNK signaling pathway is activated (Ramet, 2002).

Interestingly, puc-lacZ expression does not begin immediately after wounding. X-gal staining stays negative during the first hours after the injury. Not until 3 h after wounding is puc-lacZ expression activated. At this point, the epidermal edge expressing puc is separated from the melanized line that defines the edge of the wound. There appears to be a temporal correlation between the onset of puc gene expression and cell shape change and cell movements. By 6 h, the puc expression has become stronger and the edge of puc-lacZ expression has moved closer to the edge of the wound. The peak in puc-lacZ expression is observed about 12 h after wounding, both in terms of positive cells and staining intensity. At 18 h after wounding, puc gene expression is still observed but the number of positive cells has decreased. This may reflect reduction of cell shape change activity in epidermis. Therefore, puc gene expression in cells close to the edge of the wound is temporally associated with the cell shape change (Ramet, 2002).

In contrast to embryonic DC where only the most dorsal cell row at the leading edge is expressing puc gene, several rows of adult epidermal cells show a strong ß-galactosidase activity during wound healing. This result is consistent with high DJNK activity in the vicinity of the wound. Indeed, the extent of the area expressing puc-lacZ clearly depends on the size of the wound (up to 8 cell rows). Furthermore, puc gene expression showed a decreasing gradient from the edge of the wound towards healthy epithelium. This suggests that a newly formed signal emerges from the wound and diffuses through the epidermal layer (Ramet, 2002).

necrotic gene (nec) encodes a putative serine protease inhibitor of the serpin family. Mutant flies have necrotic areas in the epidermis of the body and the leg joints. TEM analysis has revealed the presence of necrotic epidermal cells under melanized spots. In most cases, a new layer of epidermis is observed underneath the necrotic cells, presumably replacing the dying cells. To determine whether puc gene induction is correlated to the epithelial regeneration, pucE69 P-lacZ expression was investigated in a nec mutant background. A strong ß-galactosidase staining was observed in the neighborhood of necrotic spots. puc expression is clearly induced in the epithelial region surrounding necrotic areas. puc-dependent ß-galactosidase is also observed at necrotic spots located at leg junction (Ramet, 2002).

These observations suggest that puc expression is not only induced by wounding but also by necrotic injury of the epidermis. Therefore, puc expression seems to correlate with the spreading and fusing of the two epithelia of an injured epidermis (Ramet, 2002).

During embryonic DC, the DJNK pathway activates puc expression in the LE and as a negative feedback loop, puc itself down-regulates the DJNK. To further test if the DJNK pathway also mediates puc induction during wound healing, puc expression pattern in adult epidermis was analyzed in fly mutants of this pathway. In hep, a hypomorphic allele of hemipterous, (encoding the MAPKK), puc-lacZ expression is almost completely normal. This result is not totally unexpected since hep flies present no epidermal defects. Stronger hep allele mutations are lethal and could not be studied. However, a heteroallelic combination of kay mutations leads to viable flies. Since the incidence of dorsal thoracic cleft phenotype of this mutant is higher than that in hep, it is more likely to affect the regulation of puc expression. In kay1/kay2 animals, puc gene induction is drastically reduced at the site of injury compared to wild-type. This result demonstrates that puc regulation is dependent on the transcriptional activator DFos during wound healing. Interestingly, still 18 h after wounding, the mutant cells are separated from the edge of the wound comparably to that observed with wild type at 3 h after wounding. This suggests that the epidermal sheet has been unable to spread under the wound and that the process is blocked at the initiation stage. Since DFos, together with DJun, is the target of the DJNK pathway, and forms the transcription factor AP-1, it is likely that the DJNK pathway is switched on by an integument injury (Ramet, 2002).

To find out if DFos mutation has a cell autonomous effect, the UAS/GAL4 system was used to express a dominant negative form of DFos in the pannier (pnr) expression domain. In the pnr-Gal4 line, Gal4 protein is expressed in a large dorsal band of adult epidermis. A continuous wound was done to overlap this dorsal epidermal expression domain and the dorso-lateral and ventral epidermal domain. puc-lacZ expression was then assayed 12 h after wounding. In control flies, puc is expressed at the wounded epidermis independent of the location. When DFosbZip dominant negative form of DFos is expressed in the dorsal band, X-Gal staining shows a clear, albeit not total, reduction of puc expression at the expected places. In contrast, puc expression is induced normally outside of the pnr expression domain. This demonstrates that the puc gene induction is under the control of the DFos transcriptional factor in a cell-autonomous manner similar to that observed during dorsal and thorax closure (Ramet, 2002).

To ascertain the importance of the DJNK pathway in wound healing, the phenotype of kay mutant was investigated during the course of wound healing. As expected, the wounded epidermis from kay deficient flies fails to recover. The epithelial cells at the edge of the wound also fail to undergo any evident cell shape change or show any cytoplasmic protrusive extensions. Even at 18 h after injury, the wound is not repaired. Therefore, the transcriptional activator DFos appears necessary for a normal epithelial repair in adult Drosophila. Interestingly, over a period of 6 days, wounded mutant flies did not suffer any higher mortality compared to wounded wild type flies, suggesting that epithelial repair is not crucial for early survival (Ramet, 2002).

Eiger, the first invertebrate tumor necrosis factor (TNF) superfamily ligand that can induce cell death, was identified in a large-scale gain-of-function screen. Eiger is a type II transmembrane protein with a C-terminal TNF homology domain. It is predominantly expressed in the nervous system. Genetic evidence shows that Eiger induces cell death by activating the Drosophila JNK pathway. Although this cell death process is blocked by Drosophila inhibitor-of-apoptosis protein 1 (DIAP1, Thread), it does not require caspase activity. Genetically, Eiger has been shown to be a physiological ligand for the Drosophila JNK pathway. These findings demonstrate that Eiger can initiate cell death through an IAP-sensitive cell death pathway via JNK signaling (Igaki, 2002).

Puckered (Puc) is a dual-specificity phosphatase, the expression of which is induced by the Drosophila JNK pathway to inactivate Bsk, so that puc expression can be used to monitor the extent of activation of the JNK pathway. To confirm that the JNK pathway is actually activated by Eiger, puc expression level was assessed in the eye disc of GMR>regg1GS9830 flies using a puc-LacZ enhancer-trap allele. The strong induction of puc-LacZ was observed in the region posterior to the morphogenetic furrow of the eye disc compared with the control eye disc. Furthermore, Western blot analysis with an anti-phospho-JNK antibody has revealed that Bsk is phosphorylated by Eiger overexpression. These genetic and biochemical data led to a model in which Eiger activates Msn, thereby triggering the JNK signaling pathway, sequentially stimulating dTAK1, Hep and Bsk. Using RT-PCR analysis, whether Eiger could stimulate the NF-kappaB pathway was tested; however, no obvious upregulation of the antimicrobial peptide genes, the target genes of the Drosophila NF-kappaB pathway, was detected (Igaki, 2002).

The nuclear zinc-finger protein encoded by the hindsight (hnt) locus regulates several cellular processes in Drosophila epithelia, including the Jun N-terminal kinase (JNK) signaling pathway and actin polymerization. Defects in these molecular pathways may underlie the abnormal cellular interactions, loss of epithelial integrity, and apoptosis that occurs in hnt mutants, in turn causing failure of morphogenetic processes such as germ band retraction and dorsal closure in the embryo. To define the genetic pathways regulated by hnt, 124 deficiencies on the second and third chromosomes and 14 duplications on the second chromosome were assayed for dose-sensitive modification of a temperature-sensitive rough eye phenotype caused by the viable allele, hntpeb; 29 interacting regions were identified. Subsequently, 438 P-element-induced lethal mutations mapping to these regions and 12 candidate genes were tested for genetic interaction, leading to identification of 63 dominant modifier loci. A subset of the identified mutants also dominantly modify hnt308-induced embryonic lethality and thus represent general rather than tissue-specific interactors. General interactors include loci encoding transcription factors, actin-binding proteins, signal transduction proteins, and components of the extracellular matrix. Expression of several interactors was assessed in hnt mutant tissue. Five genes -- apontic (apt), Delta (Dl), decapentaplegic (dpp), karst (kst), and puckered (puc) -- regulate tissue autonomously and, thus, may be direct transcriptional targets of Hnt. Three of these genes -- apt, Dl, and dpp -- are also regulated nonautonomously in adjacent non-Hnt-expressing tissues. The expression of several additional interactors -- viking (vkg), Cg25, and laminin-alpha (LanA) -- is affected only in a nonautonomous manner (Wilk, 2004).

Suppression of Polycomb group proteins by JNK signalling induces transdetermination in Drosophila imaginal discs

The PcG proteins function through cis-regulatory elements called PcG response elements (PREs), which enable them to bind and to maintain the state of transcriptional silencing over many cell divisions. PcG proteins operate in two key evolutionarily conserved chromatin complexes, and reduced expression of these complexes, as found in PcG mutants, results in the derepression of PRE-controlled genes. To determine whether PcG silencing is modulated in regenerating tissue, the FLW-1 line, which contains a lacZ reporter gene under the control of the Fab7 PRE, was used. Prothoracic leg discs silent for lacZ expression were fragmented and transplanted into the abdomen of host flies. Flies were fed with 5-bromodeoxyuridine (BrdU) to mark the regenerated tissue (the blastema). In uncut discs, there was little proliferation and expression of lacZ was undetectable. On fragmentation, however, lacZ was expressed in the blastema. To confirm that this derepression was due to a reduction in PcG silencing and not simply to massive proliferation at the wound site, the line LW-1 was used; this line lacks the Fab7 PRE and is normally silent, but it can be activated by induction of GAL4. Neither uncut nor cut leg discs of the LW-1 line showed expression of lacZ after transplantation (Lee, 2005).

To show that transdetermination takes place only in cells with downregulated PcG function, fragmented leg discs of the FLW-1 line were stained for lacZ expression and for Vg in order to visualize the transdetermination to wing fate. It was consistently observed that the Vg staining lay within the lacZ expression domain, suggesting that PcG genes are downregulated in the blastema, enabling PRE-silenced genes to be reactivated according to new morphogenetic cues (Lee, 2005).

To investigate direct targets of PcG regulation that, when reactivated, might contribute to transdetermination, the PREs predicted at the wg and vg genes were tested and both were found to be controlled by PcG proteins. The fact that both the transgenic vg-lacZ reporter construct (which lacks the PRE) and the endogenous vg gene were upregulated in the blastema suggests that PcG proteins may affect vg expression both indirectly (for example, through wg) and directly by means of the vg PRE (Lee, 2005).

JNK signalling in Drosophila is crucial for wound healing and is implicated in many different developmental processes, such as dorsal and thorax closure. hemipterous encodes the JNK kinase (JNKK) that activates the Drosophila JNK Basket. Products of DJun and kayak (the Drosophila homologue of Fos) form the AP-1 transcription factor. A downstream target of JNK signalling is puckered (puc), which encodes a phosphatase that selectively inactivates Basket and thus functions in a negative feedback loop. The expression of puc thus mirrors JNK activity. Because wound healing takes place after fragmentation, it was reasoned that activation of the JNK pathway might be causing the downregulation of PcG proteins in the blastema. The pucE69 line, which carries a P(lacZ) insertion at the puc locus, was used to monitor JNK activity. During the third-instar larval stage puc is not expressed and thus JNK signalling was not activated in leg discs. As expected, however, puc was expressed on fragmentation in all cells at the annealing cut edge (Lee, 2005).

To check whether cells that have activated the JNK pathway also show transdetermination, fragmented leg discs of flies carrying the puc-lacZ reporter and vgBE-Gal4; UAS-GFP constructs were transplanted. In these flies, cells that adopted a wing fate were identified by their expression of green fluorescent protein (GFP). Two days after fragmentation, weak residual puc-lacZ staining was still visible in the central region of the disc. puc-lacZ staining is known to decline rapidly after wound healing is completed. It was found that stronger staining was visible along the cut site, probably owing to ongoing wound healing. On comparison of puc-lacZ staining and GFP fluorescence, JNK-active cells showed a substantial overlap with transdetermined cells; thus, it is concluded that JNK signalling is activated in cells that undergo transdetermination (Lee, 2005).

JNK signalling affects the transcription of numerous genes, including those encoding chromatin regulating factors. Therefore whether JNK signalling can downregulate the PcG proteins required for transdetermination was examined. A constitutively active form of hep was overexpressed in UAS-hepact; hsGal4 flies by a heat-shock pulse. Activating the JNK pathway caused a downregulation of some PcG genes, such as Pc, ph-p and E(Pc). No downregulation of these genes was observed in wild-type larvae before and after heat shock, indicating that this was not an unspecific heat-shock response. Expression was examined of two genes of the Trithorax group (ash1 and brm) that function antagonistically to PcG proteins, but found no upregulation on JNK induction (Lee, 2005).

To show further that JNK has a specific effect on PcG proteins, the analogous experiment was carried out in mammalian cells. The JNK pathway can be activated in mouse embryonic fibroblasts by exposing the cells to ultraviolet light. The expression of MPh2 (mouse polyhomeotic2) was examined because this mammalian PcG gene is expressed in these cells. The expression of MPh2 was decreased on JNK induction, but after treatment with a specific JNK inhibitor it was partially restored. In addition, to show that the downregulation of PcG genes is directly controlled by AP-1, chromatin immunoprecipitation was carried out using antibodies against Fos on chromatin from UAS-hepact; hsG4 and kay1 mutant flies. Enrichment of Fos on the promoter region of ph-p was observed, but no enrichment in chromatin from flies lacking Fos. This finding suggests that AP-1 binds directly to this region to regulate negatively the transcription of ph-p (Lee, 2005).

If activation of JNK signalling in the blastema indeed leads to a downregulation of PcG genes, then impairment of the JNK pathway should result in reduced efficiency of transdetermination. The transdetermination behaviour of wild-type discs was compared with that of discs bearing mutations in the JNKK hep. The transdetermination events were classified into three categories: large regions, small regions, and no regions of transdetermination. In wild-type discs only large regions were detected. In males hemizygous for hep1 (a weak hypomorphic allele), most transplanted leg discs had large transdetermined regions; however, a substantial proportion showed only small regions of transdetermination and a few showed no transdetermination event. In flies heterozygous for hepr75 (a null allele which is hemizygous lethal), most discs showed no or only small regions of transdetermination, and large regions were rarely seen. The morphology of the regenerated discs seemed unaffected in these mutants, indicating that the decline of transdetermination efficiency was not due to inefficient wound healing (Lee, 2005).

This study has shown that PcG genes are downregulated by JNK signalling. Because many developmental regulators need to be switched, the role of PcG downregulation may be to render the cells susceptible to a change in cell identity by shifting the chromatin to a reprogrammable state. Transdetermination has been ascribed to the action of ectopic morphogens, which induce cells to activate incorrect gene cascades. Without doubt, wg and decapentaplegic signalling must be crucially involved in this process, because transdetermination does not result from any random cut but occurs preferentially when cuts are made through particular regions of the disc called 'weak points', which are regions of high morphogen. Inappropriate or overextreme downregulation of the PcG system by JNK in sensitive cells of the weak points thus may create such aberrant local patterns. Indeed, the data indicate that at least the two patterning genes, wg and vg, may be direct targets of the PcG. Notably, hyperactive Wnt signalling can also induce a switch in lineage commitment in mammals, implying that signalling pathways are a potent inducer of cell fate changes in many organisms (Lee, 2005).

Another study has shown that regenerating and transdetermining cells in the blastema have a distinct cell-cycle profile in contrast to the surrounding normal disc cells. It has been proposed that this change in cell-cycle regulation is a prerequisite for the change in cell fate. Indeed, PcG targets include genes involved in cell-cycle regulation, suggesting that this initial step is part of the complete reprogramming cascade required for the regenerating cells to achieve multipotency. Downregulation of PcG silencing by JNK seems to be a fundamental, evolutionarily conserved mechanism of cell fate change and thus may also have implications for studies of stem cell plasticity and tissue remodelling (Lee, 2005).

Targets of Activity

During the second half of embryogenesis, decapentaplegic is expressed in specific regions in the embryo, particularly in the dorsal-most epidermal cells that express puc. It has been shown recently that the maintenance of decapentaplegic expression along the leading edge of the epidermis depends on the activity of the DJNK pathway (Glise, 1997; Hou, 1997, and Riesgo-Escovar, 1997). In pucE69 mutant embryos, the expression of dpp in the dorsal-most epidermal cells is enhanced from stage 11. Furthermore, after germ-band shortening there are more epidermal cells expressing dpp than in wild type, but this expression is still mainly limited to dorsal cells. In contrast, ubiquitous expression of Puc leads to a decrease in the expression of dpp in those cells during stage 11 and the complete absence at stages 12 and 13. Puc overexpression does not affect the levels of dpp on the visceral mesoderm or the ventral epidermis. These results suggest a role for puc in the control of dpp expression, which could be mediated by Puc activity on JNK signaling (Martin-Blanco, 1998).

The null phenotype for dpp, a completely ventralized embryo, reflects its initial role over the dorsal epidermis and might obscure the function of later patterns of expression. Mutants for thick veins, however, which encodes a Dpp receptor, display dorsal holes similar to those of hep or bsk mutants (Affolter, 1994). To test if the higher levels of dpp present in puc mutants could be involved in puckering phenotypes, dpp was overexpressed. Such dpp overexpressing embryos undergo an extreme dorsalization of the epidermis, but they still have a dorsal midline; in many cases, phenotypes were observed that were very similar to those observed in pucE69 mutants. This indicates that besides an early function for dpp in epidermal cell stretching, the downregulation of dpp in the dorsal-most epidermal cells is necessary for completion of dorsal closure (Martin-Blanco, 1998).

Two Drosophila tumor necrosis factor receptor-associated factors (TRAFs), Traf1 and Traf2, are proposed to have similar functions with their mammalian counterparts as a signal mediator of cell surface receptors. However, as yet their in vivo functions and related signaling pathways are not fully understood. Traf1 is shown to be an in vivo regulator of c-Jun N-terminal kinase (JNK) pathway in Drosophila. Ectopic expression of Traf1 in the developing eye induces apoptosis, thereby causing a rough-eye phenotype. Further genetic interaction analyses reveal that the apoptosis in the eye imaginal disc and the abnormal eye morphogenesis induced by Traf1 are dependent on JNK and its upstream kinases, Hep and TGF-ß activated kinase 1. In support of these results, the Traf1-null mutant shows a remarkable reduction in JNK activity with an impaired development of imaginal discs< and a defective formation of photosensory neuron arrays. In contrast, Traf2 was demonstrated as an upstream activator of nuclear factor-kappaB (NF-kappaB). Ectopic expression of Traf2 induces nuclear translocation of two Drosophila NF-kappaBs, Dif and Relish, consequently activating the transcription of the antimicrobial peptide genes diptericin, diptericin-like protein, and Drosomycin. Consistently, the null mutant of Traf2 shows immune deficiencies in which NF-kappaB nuclear translocation and antimicrobial gene transcription against microbial infection were severely impaired. Collectively, these findings demonstrate that Traf1 and Traf2 play pivotal roles in Drosophila development and innate immunity by differentially regulating the JNK- and the NF-kappaB-dependent signaling pathway, respectively (Cha, 2003).

To investigate the consequences of ectopic expression of Traf1 in the developing Drosophila eye, Traf1 was overexpressed by using an eye-specific gmr-GAL4 driver. The eyes of adults carrying one copy each of both gmr-GAL4 and Traf1 show a rough-eye surface with disorganized arrays of ommatidia, whereas the eyes of flies carrying either one copy of gmr-GAL4 or one copy of Traf1 alone appear normal. Examination of the retinal sections of adults carrying both gmr-GAL4 and Traf1 reveals the number of ommatidia to be reduced and the number and shape of the photoreceptor cells in each ommatidium also to be abnormal, compared to the controls, which carry only the gmr-GAL4 driver (Cha, 2003).

When two copies of Traf1 were overexpressed in the eye, it displayed a more severe phenotype and a reduced number of ommatidia, resulting in a size reduction of the compound eye, and some ommatidia were fused with each other. In contrast, ectopically expressed Traf2 had no effect on the eye development; ommatidial array, bristles, and compound eye size were all found to be normal (Cha, 2003).

In order to further confirm that Traf1 activates the JNK kinase signaling pathway at a molecular level, JNK activity in the eye discs was examined by two different experimental approaches -- an immunohistochemical assay with anti-phospho-specific JNK antibody and a puckered-LacZ reporter assay. JNK phosphorylation is highly induced by overexpression of Traf1 compared to the control. In addition, expression of puckered (puc), a well-known downstream target of JNK, is also highly induced by Traf1 compared to the control. Collectively, the results clearly demonstrated that Traf1 activates the JNK signaling pathway in vivo (Cha, 2003).

An RNA interference screen identifies Inhibitor of Apoptosis Protein 2 as a regulator of innate immune signalling in Drosophila: IAP2 acts upstream of puckered

Innate immunity in vertebrates and invertebrates is of central importance as a biological programme for host defence against pathogenic challenges. To find novel components of the Drosophila immune deficiency (IMD) pathway in cultured haemocyte-like cells, RNA interference library was screened for modifiers of a pathway-specific reporter. Selected modifiers were further characterized using an independent reporter assay and placed into the pathway in relation to known pathway components. Interestingly, the screen identified the Inhibitor of apoptosis protein 2 (IAP2) as being required for IMD signalling. Whereas loss of DIAP1, the other member of the IAP protein family in Drosophila, leads to apoptosis, IAP2 is dispensable for cell viability in haemocyte-like cells. Cell-based epistasis experiments show that IAP2 acts at the level of Tak1 (transforming growth factor-ß-activated kinase 1). The results indicate that IAP gene family members may have acquired other functions, such as the regulation of the tumour necrosis factor-like IMD pathway during innate immune responses (Gesellchen, 2005).

The results indicated that IAP2 is required for the IMD-Rel branch and cell-based epistasis mapped it downstream of IMD and upstream of Relish. Since the IMD pathway branches at the same level or downstream of Tak1 into Rel- and JNK-dependent signalling, whether IAP2 is also required for activation of the JNK branch was examined. Thus, the expression of the IMD/JNK-specific target genes Puckered and Matrix metalloproteinase 1 (Mmp-1) was examined by qPCR. Depletion of IAP2 by RNAi disrupts Mmp-1 and puc induction after innate immune stimuli to a level similar to that of knockdown of known factors specific for the IMD-JNK branch (Mkk4/hep). These experiments, together with the epistasis experiments, support a model whereby IAP2 acts, similarly to Tak1, downstream of IMD and upstream or at the level of the branching point of the IMD-Rel and IMD-JNK signalling arms (Gesellchen, 2005).

This study has identified several new components of the IMD innate immune pathway. The experiments implicate several signalling factors in the control of IMD-dependent responses in haemocyte-like cells, including a GTPase-activating protein, a homologue of the mammalian Tak1-binding protein, and several proteins involved in RNA binding and processing. Their role in Drosophila immune response in vivo remains to be characterized. Strikingly, the screen identifies IAP2, a member of the Inhibitor of Apoptosis Protein family, as being required for Drosophila innate immune signalling. IAP2 is specifically involved in the IMD signalling pathway, since it disrupts the induction of the IMD-Rel and IMD-JNK pathway target genes and is not required for other immune-induced pathways, such as Toll and JAK-STAT. Cell-based epistasis analysis and qPCR experiments monitoring the IMD-JNK branch suggest a function of IAP2 downstream of IMD and upstream or at the same level as Tak1. Although most previously characterized IAPs were shown to act as inhibitors of caspases, it is unlikely that the role of IAP2 is to inhibit DREDD, the caspase implicated in IMD signalling. If this were correct, depletion of IAP2 should lead to an enhancement of pathway activity after immune stimulus or to a constitutive expression of target genes without an immune stimulus, which is not the case. Since human Tak1 has been shown to be activated by polyubiquitination, and it has recently been shown that ubiquitination is required for the activation of Tak1 and the IKK complex in Drosophila, it was speculated that IAP2 may have a role in Tak1 ubiquitination through its RING domain. Whether mammalian IAPs have a role in innate immune responses remains to be established (Gesellchen, 2005).

Protein Interactions

The mutation puckered was isolated by random insertion of a transcriptional activator into Drosophila genes. One insertion site inactivates puckered, creating a mutation that results in an abnormal morphology of dorsal epidermal cells (Ring, 1993). In spite of this abnormal morphology, dorsal closure occurs to completion The puckered gene is not expressed in hemipterous mutants, indicating that Puckered acts as a downstream target of hemipterous, and that one function of hep is to activate genes expressed in cells involved in dorsal closure. Although the function of puckered is not known, discovery of a target of HEP provides a hint as to the existence of a dorsal closure pathway (Glise, 1995).

Evidence has been found that Puckered is a JNK specific phosphatase. When extracts from wild-type and puc mutant embryos are assayed for their endogenous JNK activity, puc mutants show a significant increase in JNK activity, when compared with wild type. Wild-type embryos have high levels of JNK phosphatase activity. Puc extracts do not show JNK phosphatase activity. Extracts prepared from puc mutant embryos showed a twofold increase in JNK activity relative to wild type. In hep extracts, JNK phosphatase activity is reduced to 50% of that of wild-type embryos. When similar extracts are tested for their ability to inactivate preactivated JNK, up to 50% inhibition is obtained after 30 min from wild-type embryos, as compared with abolition of activity in extracts from puc mutants (Martin-Blanco, 1998).

If puc encodes a JNK phosphatase activity, then it should inactivate JNK in vivo. To test if this is the case, the puc cDNA was ectopically expressed. Ubiquitous expression of Puc results in a dorsal hole during embryogenesis reminiscent of the phenotype of mutations in hep and bsk that encode a JNKK and a JNK, respectively (Martin-Blanco, 1998).

During Drosophila embryogenesis, Jun kinase (JNK) signaling has been shown to play a key role in regulating the morphogenetic process of dorsal closure, which also serves as a model for epithelial sheet fusion during wound repair. During dorsal closure the JNK signaling cascade in the dorsal-most (leading edge) cells of the epidermis activates the AP-1 transcription factor comprised of Jun and Fos that, in turn, upregulates the expression of the dpp gene. Dpp is a secreted morphogen that signals lateral epidermal cells to elongate along the dorsoventral axis. The leading edge cells contact the peripheral cells of a monolayer extraembryonic epithelium, the amnioserosa, which lies on the dorsal side of the embryo. Focal complexes are present at the dorsal-most membrane of the leading edge cells, where they contact the amnioserosa. The JNK signaling cascade is initially active in both the amnioserosa and the leading edge of the epidermis. JNK signaling is downregulated in the amnioserosa, but not in the leading edge, prior to dorsal closure. The subcellular localization of Fos and Jun is responsive to JNK signaling in the amnioserosa: JNK activation results in nuclear localization of Fos and Jun; the downregulation of JNK signaling results in the relocalization of Fos and Jun to the cytoplasm. The Hindsight (Hng) Zn-finger protein and the Puckered (Puc) JNK phosphatase are essential for downregulation of the JNK cascade in the amnioserosa. Persistent JNK activity in the amnioserosa leads to defective focal complexes in the adjacent leading edge cells and to the failure of dorsal closure. Thus focal complexes are assembled at the boundary between high and low JNK activity. In the absence of focal complexes, miscommunication between the amnioserosa and the leading edge may lead to a premature 'stop' signal that halts dorsalward migration of the leading edge. Spatial and temporal regulation of the JNK signaling cascade may be a general mechanism that controls tissue remodeling during morphogenesis and wound healing (Reed, 2001).

The transcriptional activation of the genes dpp and puc provides a readout of JNK signaling activity in the leading edge. Enhancer trap lines dpplacZ and puclacZ were used as reporters for the activation state of the JNK pathway in the amnioserosa. These enhancer trap lines are expressed in the amnioserosa prior to germ band retraction. Toward the end of germ band retraction, JNK activity, as assayed by puclacZ and dpplacZ, decreases in the interior of the amnioserosa but persists in the amnioserosa perimeter cells that abut the leading edge. By the onset of dorsal closure, when JNK activity becomes elevated in the leading edge, the amnioserosa perimeter cells lose JNK activity, and there is reduced dpplacZ or puclacZ expression throughout the amnioserosa. It should be noted that perdurance of ß-galactosidase protein in the amnioserosa means that these analyses of the timing of loss of puclacZ and dpplacZ expression define the latest point in development at which JNK signaling is downregulated, not when such downregulation initiates. It is concluded that JNK signaling occurs in the amnioserosa prior to and during germ band retraction but is downregulated at or before the initiation of dorsal closure (Reed, 2001).

DJUN is activated through phosphorylation by JNK, and although it is capable of forming transcriptional activation complexes through homodimerization, it also forms heterodimers with Fos. Jun/Fos heterodimers belong to the AP-1 class of transcription factor complexes, are more stable than Jun homodimers, and are thought to be the biologically relevant protein complex (Reed, 2001).

To further investigate JNK signaling in the amnioserosa during dorsal closure, the expression of Jun and Fos were examined. In wild-type embryos, Jun and Fos accumulate at high levels in the amnioserosa prior to dorsal closure. During dorsal closure, Jun and Fos levels are highest in the leading edge but persist in the amnioserosa. In the amnioserosa, Jun and Fos are strictly nuclear prior to germ band retraction. Strikingly, both proteins begin to accumulate in the cytoplasm as germ band retraction is completed. While Fos becomes nearly exclusively cytoplasmic, Jun can be detected in both the cytoplasm and the nuclei during dorsal closure (Jun is present in a punctate pattern in the cytoplasm) (Reed, 2001).

To determine whether nuclear restriction of Jun and Fos is dependent on JNK signaling, Jun and Fos expression and subcellular localization were examined in genetic backgrounds that are either reduced or elevated with respect to JNK signaling. In bsk2 embryos, which are deficient in JNK activity, the amnioserosal cells show strong cytoplasmic localization of Jun and Fos. The cytoplasmic localization is clearly enhanced in bsk2/+ embryos, relative to wild-type, suggesting that nuclear versus cytoplasmic localization of Jun and Fos is particularly sensitive to reduction in JNK signaling levels. To test the effect of increasing JNK activity in the amnioserosa, puc mutant embryos were immunostained. In this background, JNK activity is upregulated, and both Jun and Fos were restricted to the nuclei of the amnioserosal cells throughout embryogenesis. This is the first report of nucleo-cytoplasmic regulation of Jun and Fos localization in Drosophila in response to JNK signaling. Jun and Fos nuclear localization as well as dpplacZ and puclacZ expression support the conclusion that JNK signaling occurs in the amnioserosa prior to dorsal closure. Reciprocally, the reduction of dpplacZ and puclacZ expression and the movement of Jun and Fos from the nucleus into the cytoplasm of amnioserosal cells are indicative of downregulation of JNK signaling in this tissue prior to and during dorsal closure (Reed, 2001).

Given the phenotypic similarities between hnt and JNK signaling mutants, the genetic interactions between hnt and the JNK pathway mutants and observations that JNK signaling is normally downregulated in the amnioserosa prior to dorsal closure, it was asked whether molecular confirmation for Hnt as a negative regulator of JNK signaling could be found (Reed, 2001).

Hnt is not required for Jun and Fos expression, since these proteins are present in the amnioserosa of hnt mutant embryos at levels roughly comparable to wild-type. Strikingly, in contrast to wild-type embryos, hnt mutant embryos (hnt308 and hntXO01) show persistent nuclear localization of Jun and Fos. These results are consistent with the postulated role of Hnt as a negative regulator of JNK signaling in the amnioserosa (Reed, 2001).

Persistent nuclear localization of Jun and Fos is seen, not only in the amnioserosa of hnt mutants, but also in the amnioserosa of puc mutants in which dorsal closure also fails. Thus hnt and puc mutants provide independent lines of evidence that downregulation of JNK signaling in the amnioserosa is essential for dorsal closure (Reed, 2001).

Formation or maintenance of focal complexes in the leading edge of the epidermis is disrupted by persistent JNK signaling in the amnioserosa. In wild-type embryos, phosphotyrosine and F-actin accumulate conspicuously along the dorsal-most leading edge cell membranes that abut the amnioserosa, representing focal complexes. Focal complexes fail to accumulate in leading edge cells of puc mutants. Similarly, in hnt mutants, phosphotyrosine and F-actin fail to accumulate at the dorsal-most membrane of the leading edge cells. Thus, Hnt function in the amnioserosa is necessary for the adjacent leading edge cells to assemble or maintain focal complexes at their dorsal-most membranes (Reed, 2001).

The failure of focal complex assembly in the leading edge cells of hnt and puc mutants is not a secondary consequence of the failure of JNK signaling in these cells. This conclusion derives from the fact that dpplacZ and puclacZ are expressed in the leading edge of wild-type, puc, and hnt mutants. Consistent with this result, the cells of the lateral epidermis undergo dorsal-ventral elongation in puc and hnt mutants, a process dependent on JNK-induced signals from the leading edge (Reed, 2001).

The simplest interpretation of these data is that assembly or maintenance of focal complexes in the leading edge occurs only if there is a boundary between high and low JNK signaling at the junction of the leading edge (high) and the amnioserosa (low). In hnt and puc mutants, since JNK signaling persists in the amnioserosa, such a high/low JNK activity boundary never forms, and therefore focal complexes are either not assembled or are not maintained at the dorsal membrane of the leading edge. In the absence of focal complexes, the leading edge is unable to move over the amnioserosa (Reed, 2001).

The hypothesis that focal complexes form only when there is a high/low JNK signaling boundary at the juxtaposition of the leading edge and the amnioserosa predicts that conversion of a high/high back to a high/low condition will lead to the restoration of focal complexes. Therefore JNK activity was downregulated in the amnioserosa of hnt mutants during the stages at which JNK signaling would abnormally persist. This was accomplished by expressing Puc or dominant-negative JNK using an amnioserosa-specific GAL4 driver. In hnt mutants (the high/high situation), focal complexes are absent from the leading edge, and the morphology of the leading edge cells is highly abnormal. Consistent with the hypothesis, when either Puk or dominant-negative JNK is expressed in the amnioserosa of hnt mutants, focal complexes are restored to the dorsal-most membrane of the leading edge, and the morphology of the leading edge is shifted toward wild-type (Reed, 2001).

In summary, these analyses show that focal complexes fail to accumulate in the leading edge when there is no JNK signaling boundary between the leading edge and the amnioserosa. The restoration of a high/low situation by the expression of either Puc or dominant-negative JNK in a hnt mutant results in the restoration of focal complexes in the leading edge (Reed, 2001).

puckered: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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