basket/JNK

Proteins acting upstream of Basket/JNK

The small GTPases Rac and Cdc42 have been implicated together with JNKK and JNK in the JNK pathway in vertebrates (Coso, 1995 and Minden, 1995). In Drosophila the homologs have been cloned (Dcdc42 and Drac), but no mutations have been isolated (Eaton, 1995). Expression of a dominant negative form of DracA results in dorsal closure phenotypes (Harden, 1995). A dominant negative form of Dcdc42 also shows a dorsal open phenotype. This result suggests that Dcdc42 like DracA acts in dorsal closure regulation (Riesgo-Escovar, 1996).

Recombinant Hemipterous was tested for its ability to phosphorylate and activate DJNK. DJNK acts as a substrate for HEP, providing evidence for a signal transduction pathway between HEP and DJNK. DJUN is also phosphorylated in the presence of HEP and DJNK, but not in the presence of HEP alone. Lipopolysaccharide also serves to stimulate DJUN phosphorylation, suggesting that LPS is able to stimulated the HEP-DJNK-DJUN pathway (Sluss, 1996).

Cdc42 and Rac1 are required for a diverse set of cytoskeleton-membrane interactions in different cell types. These two proteins contribute differently to the organization of epithelial cells in the Drosophila wing imaginal disc. Drac1 is required to assemble actin at adherens junctions. Failure of adherens junction actin assembly in Drac1 dominant-negative mutants is associated with increased cell death. In contrast, Dcdc42 is required for processes that involve polarized cell shape changes during both pupal and larval development. In the third larval instar, Dcdc42 is required for apico-basal epithelial elongation. Whereas normal wing disc epithelial cells increase in height more than twofold during the third instar, cells that express a dominant-negative version of Dcdc42 remain short and are abnormally shaped. Dcdc42 localizes to both apical and basal regions of the cell during these events, and mediates elongation, at least in part, by effecting a reorganization of the basal actin cytoskeleton. These observations suggest that a common cdc42-based mechanism may govern polarized cell shape changes in a wide variety of cell types (Eaton, 1995).

DPAK is a Drosophila homolog of the serine/threonine kinase PAK, a protein that is a target of the Rho subfamily proteins Rac and Cdc42. Rac, Cdc42, and PAK have previously been implicated in signaling by c-Jun amino-terminal kinases. DPAK binds to activated (GTP-bound) Drosophila Rac (DRacA) and Drosophila Cdc42. Similarities in the distributions of DPAK, integrin, and phosphotyrosine suggested an association of DPAK with focal adhesions and Cdc42- and Rac-induced focal adhesion-like focal complexes. DPAK expression is elevated in the leading edge of epidermal cells, the same cells whose morphological changes drive the dorsal closure of the embryo. The accumulation in these cells of cytoskeletal elements initiating cell shape changes can be inhibited by expression of a dominant-negative DRacA transgene. Leading-edge epidermal cells flanking segment borders, which express particularly large amounts of DPAK, undergo transient losses of cytoskeletal structures during dorsal closure. DPAK may be regulating the cytoskeleton through its association with focal adhesions and focal complexes and/or may be participating with DRacA in a c-Jun amino-terminal kinase signaling pathway recently demonstrated to be required for dorsal closure (Harden, 1996).

Two Drosophila genes, DRacA and DRacB, encode proteins with homology to mammalian Rac1 and Rac2. Transgenic flies were created bearing dominant inhibitory (N17DRacA), and wild-type versions of the DRacA cDNA under control of an Hsp70 promoter. Expression of the N17DRacA transgene during embryonic development causes a high frequency of defects in dorsal closure, due to disruption of cell shape changes in the lateral epidermis. Embryonic expression of N17DRacA also affects germband retraction and head involution. The epidermal cell shape defects caused by expression of N17DRacA are accompanied by disruption of a localized accumulation of actin and myosin, two proteins thought to drive epidermal cell shape change (Harden, 1995).

Rac activation is thought to be important for stimulating dorsal closure because expression of dominant negative forms of Rac (DN Rac) or Cdc42 inhibits dorsal closure in the Drosophila embryo. Since activated Jra/Djun rescues the defect in dorsal closure induced by expression of DN Rac, Rac probably functions upstream of JNK activation to stimulate dorsal closure. To begin to address the mechanism whereby Rac and Misshapen cooperate to activate JNK, cultured cells were transfected with either Msn or NIK, together with DN Rac and an epitope-tagged JNK, and kinase activity assays were performed on JNK precipitates. Although overexpression of either NIK or msn leads to a four- to five-fold increase in JNK activation, coexpression of DN Rac markedly decreases JNK activation. Because PAK family Ste20 kinases are activated by GTP-bound Cdc42 and Rac, it had been assumed that this family of Ste20 kinases rather than an SPS1 Ste20 kinase family member would cooperate with Rac to activate JNK. Thus, discovery of the role of Misshapen in conveying Rac signals to JNK has stimulated consideration of new paradigms for how Rac functions to activate JNK. It is not thought that Rac activates Msn directly. Unlike PAK family members, Msn does not contain a consensus Rac-binding motif and no binding of Msn to activated Rac in vitro can be detected. Rather, it is hypothesized that Rac cooperates with Msn to activate a downstream MKKK. MKKKs have been shown to bind GTP-bound Cdc42 or Rac. Thus, Rac may cooperate with Msn to regulate a downstream MKKK in a manner similar to the way Ras cooperates with a yet to be defined kinase to activate RAF. In this model, binding of an MKKK to activated Rac would facilitate interaction of this MKKK with Msn, thereby enabling its activation by Msn. However, the possibility cannot be excluded that Rac and Msn activate parallel pathways converging on JNK activation (Su, 1998).

The role of DJNK in dorsal closure is to phosphorylate and activate Djun, resulting in transcriptional activation of the dpp gene at the leading edge of the dorsal epidermis. In turn, the binding of Dpp to its receptors Thick veins (Tkv) and Punt (Put) on the ventrally adjacent epithelial cells induces reorganization of the cytoskeleton, leading to epithelial cell elongation and subsequent closure over the amnioserosa. This is supported by the findings that dpp expression is decreased in the dorsal-most epithelial cells in embryos lacking bsk and hep, and that expression of activated forms of Djun or Tkv rescues embryos zygotically mutant for bsk or Jra. Therefore, if msn functions to initiate dorsal closure by activating Basket/JNK, dpp expression in the leading edge epithelial cells should be decreased in msn mutant embryos. In agreement with the idea that msn functions upstream of bsk, dpp expression in leading edge cells surrounding the amnioserosa is observed to be decreased in embryos lacking msn to a degree comparable to bsk¹ embryos. About 20% of embryos derived from the msn¹⁰²/+; msn¹⁰²/+ cross display a decrease in dpp expression in the dorsal leading edge. The decrease in dpp staining in msn mutant embryos is limited to the dorsal rim cells (Su, 1998 and references)

Two families of protein kinases that are closely related to Ste20 in their kinase domain have been identified: the p21-activated protein kinase (Pak) and SPS1 families. In contrast to Pak family members, SPS1 family members do not bind and are not activated by GTP-bound p21Rac and Cdc42. A member of the SPS1 family, called Misshapen (Msn), has been placed genetically upstream of the c-Jun amino-terminal (JNK) mitogen-activated protein (MAP) kinase module in Drosophila. The failure to activate JNK in Drosophila leads to embryonic lethality due to the failure of these embryos to stimulate dorsal closure. Msn probably functions as a MAP kinase kinase kinase kinase in Drosophila, activating the JNK pathway via an as yet undefined MAP kinase kinase kinase. A Drosophila TNF-receptor-associated factor, DTRAF1, has been identified by screening for Msn-interacting proteins using the yeast two-hybrid system. In contrast to the mammalian TRAFs that have been shown to activate JNK, DTRAF1 lacks an amino-terminal 'Ring-finger' domain, and overexpression of a truncated DTRAF1, consisting of only its TRAF domain, activates JNK. Another DTRAF, DTRAF2, has been identified that contains an amino-terminal Ring-finger domain. Msn specifically binds the TRAF domain of DTRAF1 but not that of DTRAF2. The region between the kinase and C-terminal regulatory domains of Msn is sufficient to bind DTRAF1, whereas neither the kinase domain nor the C-terminal domain alone can bind the protein. A stretch of about 250 amono acids that lies in the N-terminal portion of the interdomain is sufficient for Msn to bind DTRAF1. The C-terminal portion of this region does not interact with DTRAF1 but does interact with the SH3 domains of Dreadlocks, supporting the idea that the central region couples Msn and related Ste20 kinases to multiple upstream targets. DTRAF1 and DTRAF2 can dimerize with themselves but cannot form heterodimers. In Drosophila, DTRAF1 is thus a good candidate for an upstream molecule that regulates the JNK pathway by interacting with, and activating, Msn. Consistent with this idea, expression of a dominant-negative Msn mutant protein blocks the activation of JNK by DTRAF1. Furthermore, coexpression of Msn with DTRAF1 leads to the synergistic activation of JNK. A truncated Msn lacking the kinase domain functions as a dominant-negative inhibitor by blocking activation of JNK by DTRAF1. Some of these observations are extended to the mammalian homolog of Msn, Nck-interacting kinase (NIK), suggesting that TRAFs also play a critical role in regulating Ste20 kinases in mammals (Liu, 1999).

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 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).

Expression of the Hnt Zn-finger transcription factor in the amnioserosa, particularly in those cells that abut the leading edge of the epidermis, is essential for this morphogenetic process. Hnt function has been shown in this study to be necessary for dorsal closure. A subset of hnt mutant embryos carrying the embryonic lethal alleles hnt^704a and hnt^XO01 successfully complete germ band retraction but do not hatch. Analyses of cuticle preparations have revealed that 60% of hnt^704a and 79% of hnt^XO01 embryos that complete retraction exhibit an anterior-open or dorsal-hole phenotype characteristic of the failure of dorsal closure (Reed, 2001).

hnt and JNK signaling pathway mutants interact genetically. hnt³⁰⁸ single mutants exhibited 41% embryonic lethality. When the dose of the JNK-encoding gene, basket (bsk), is reduced in a hnt³⁰⁸ mutant background (embryonic lethality was suppressed approximately 2-fold). These results suggest that, in hnt mutants, JNK signaling is upregulated (i.e., that the function of Hnt in dorsal closure is to downregulate JNK signaling). Thus, the reduction of JNK function is able to partially suppress the dorsal closure defect in hnt³⁰⁸ mutants (Reed, 2001).

To further test the role of Hnt in regulating JNK signaling, genetic interactions between hnt³⁰⁸ and dpp mutants were examined. A 50% reduction of dpp gene dose led to an 8-fold reduction in hnt³⁰⁸ embryonic lethality (5% versus 41%). Conversely, increasing the dose of the wild-type dpp gene from two to three copies led to a 2-fold increase in embryonic lethality (80% versus 41%). Examination of the hnt³⁰⁸ embryos carrying three doses of dpp revealed that the frequency of embryos with germ band retraction defects had doubled (41% as compared to 20%). These results provide the first evidence that Hnt may regulate both germ band retraction and dorsal closure through the JNK/DPP signaling pathways. The direction of the genetic interaction between hnt³⁰⁸ and dpp is consistent with the hypothesized role of Hnt to downregulate JNK signaling (Reed, 2001).

During normal development, JNK activity is downregulated in the amnioserosa prior to dorsal closure. Hnt is expressed in the amnioserosa, but not in the epidermis of the embryo. Given the genetic interactions between hnt and JNK pathway mutants, it was therefore asked whether JNK signaling occurs in the amnioserosa during normal embryogenesis. JNK signaling is shown to initially be present in the amnioserosa but it is is downregulated prior to dorsal closure (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 dpp^lacZ and puc^lacZ 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 puc^lacZ and dpp^lacZ, 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 dpp^lacZ or puc^lacZ 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 puc^lacZ and dpp^lacZ 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 dpp^lacZ and puc^lacZ expression support the conclusion that JNK signaling occurs in the amnioserosa prior to dorsal closure. Reciprocally, the reduction of dpp^lacZ and puc^lacZ 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 hnt^XO01) 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 dpp^lacZ and puc^lacZ 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).

The small GTPase Rho is a molecular switch that is best known for its role in regulating the actomyosin cytoskeleton. Its role in the developing Drosophila embryonic epidermis during the process of dorsal closure has been investigated. By expressing the dominant negative DRhoA^N19 construct in stripes of epidermal cells, it has been confirmed that Rho function is required for dorsal closure and it is necessary to maintain the integrity of the ventral epidermis. Defects in actin organization, nonmuscle myosin II localization, the regulation of gene transcription, DE-cadherin-based cell-cell adhesion and cell polarity underlie the effects of DRhoA^N19 expression. Furthermore, these changes in cell physiology have a differential effect on the epidermis that is dependent upon position in the dorsoventral axis. In the ventral epidermis, cells either lose their adhesiveness and fall out of the epidermis or undergo apoptosis. At the leading edge, cells show altered adhesive properties such that they form ectopic contacts with other DRhoA^N19-expressing cells (Bloor, 2002).

RhoA^N19 expression causes ectopic activation of the JNK pathway in the lateral epidermis, suggesting that RhoA normally functions to inhibit JNK signaling. JNK activation is antagonized by the protein phosphatase encoded by puc, and in puc mutants JNK signaling is increased at the leading edge and is activated in the lateral epidermis. Thus, JNK signaling in wild-type embryos is not maximal and basal JNK activity in the lateral epidermis is revealed in the absence of either puc or RhoA mediated repression. Interestingly, RhoA^N19 expression does not increase JNK signaling in the leading edge. This difference between the effect of puc mutations and RhoA^N19 expression could be due to ectopic RhoA^N19 suppressing an upstream JNK activator that is itself maximally activated in the leading edge (Bloor, 2002).

Tensin is an actin-binding protein that is localized in focal adhesions. At focal adhesion sites, tensin participates in the protein complex that establishes transmembrane linkage between the extracellular matrix and cytoskeletal actin filaments. Even though there have been many studies of tensin as an adaptor protein, the role of tensin during development has not yet been clearly elucidated. Thus, this study was designed to dissect the developmental role of tensin by isolating Drosophila tensin mutants and characterizing its role in wing development. The Drosophila tensin loss-of-function mutations results in the formation of blisters in the wings, that are due to a defective wing unfolding process. Interestingly, by¹ -- the mutant allele of the gene blistery (by) -- also shows a blistered wing phenotype, but fails to complement the wing blister phenotype of the Drosophila tensin mutants, and contains Y62N/T163R point mutations in Drosophila tensin coding sequences. These results demonstrate that by encodes Drosophila Tensin protein and that the Drosophila tensin mutants are alleles of by. Using a genetic approach, it has been demonstrated that Tensin interacts with integrin and also with the components of the JNK signaling pathway during wing development; overexpression of by in wing imaginal discs significantly increases JNK activity and induces apoptotic cell death. Besides the defects in wing cell adhesion process, another distinct mutant phenotype was observed in the by mutants; they lay rounded eggs due to defective oocyte elongation during oogenesis. Collectively, these data suggest that Tensin relays signals from the extracellular matrix to the cytoskeleton through interaction with integrin, and through the modulation of the JNK signal transduction pathway during Drosophila wing development (Lee, 2003).

Drosophila wing development after pupariation (AP) consists of two distinct stages: prepupal and pupal wing morphogenesis. Pupal wing morphogenesis is further divided into three stages: separation (11-12 hours AP) of the ventral cell layer from the dorsal layer, re-apposition of the inter-vein cells (21-36 hours AP) and re-separation (60 hours AP) of the two cell layers. Shortly after eclosion, wings expand and unfold by an influx of hemolymph. PS integrins are required for the attachment of the two wing surfaces during pupal wing re-apposition and for the maintenance of the wing bilayer (Lee, 2003).

To determine the detailed roles of tensin during wing morphogenesis, the pupal wings of the by² flies were examined. No differences were observed in the attachment of two wing surfaces and in the integrin localization between wild type and by² wings during both prepupal apposition (4-6 hours AP) and pupal reapposition stages (30-36 hours AP) (Lee, 2003).

Because the pupal wing development was not disturbed in the by² mutants, the expansion and unfolding processes of adult wings in the by² mutants was investigated. After eclosion, the by² flies display folded wings similar to the controls. Then, a sudden and rapid influx of hemolymph induces the unfolding of folded wings in the by² mutants. However, as soon as the wings of by² flies unfold, fluid-filled blisters began to appear at the distal part of the wings, and the boundary of the blisters expand to a certain extent. After the fluid dries, the wing blisters are fixed in place. Taken together, although the dorsal and ventral layers of a wing can be brought into close association during apposition and re-apposition processes in the by² flies, the link between them may not be strong enough to resist the hydrostatic pressures during the wing unfolding process (Lee, 2003).

The functional significance of each domain of tensin in normal wing development was examined. UAS lines overexpressing either full-length tensin protein or various deletion mutant forms of tensin were generated. Unlike DeltaN and DeltaC, overexpression of DeltaPTB by MS1096-GAL4 driver completely rescues the blistered wing phenotype of by². These data suggest that both the N-terminal region and the SH2 domain of tensin are required for proper attachment of two wing surfaces (Lee, 2003).

Since mammalian tensin is known to participate in the integrin signaling, whether tensin genetically interacts with integrin was examined. As expected, the blistered wing phenotype become more severe in the if³; by²/+ mutants and extremely severe in the double homozygotic mutants for if³ and by², compared with if³ homozygotes or by² heterozygotes. In addition, the rate of flies showing blistered wings in the total population greatly increases in the double mutants (Lee, 2003).

In mammalian cells, tensin has been implicated in signal transduction related to cell adhesion such as Src, JNK and PI3K. To examine the role of tensin in the signaling processes related to wing development, the in vivo interaction between tensin and signaling molecules including rl/Erk, Src, JNK and PI3K was investigated. Interestingly, it was found that the JNK signaling pathway is tightly correlated with tensin in the wing development, while other signaling molecules including rl/Erk do not show any interactions with tensin. Homozygous by² mutants with heterozygotic mutations of the JNK signaling components bsk¹ or hep¹ (the loss-of-function mutants for Drosophila JNK and MKK7, respectively) display a highly severe blistered wing phenotype, compared with either homozygous by², heterozygous bsk¹ or heterozygous hep¹ mutants. Notably, the rate of flies, which show Class II blistered wings, increases from 46.5% to 70% for these double mutants compared with homozygous by² mutants, and about 15% of these flies had multiple blisters in their wings. Furthermore, the double homozygotic mutants for by² and hep¹ die at pharate adult stage. The lethality of these double mutants may be due to an impairment of essential in vivo interactions between tensin and the JNK signaling pathway in Drosophila (Lee, 2003).

Next, whether overexpressed by also interacts with the components of the JNK signaling pathway was tested. Overexpression of by using MS1096-GAL4 driver turns the adult wings into a convex shape with a smaller overall size, and this phenotype becomes more severe when two copies of the by gene are overexpressed. Simultaneous overexpression of bsk or hep with by results in a severely curled wing phenotype, which is fully penetrant, whereas overexpression of bsk or hep alone by MS1096-GAL4 driver did not induce any detectable phenotypes in the wing. Collectively, these data suggest that tensin activity is highly related to the JNK signaling pathway during wing development in Drosophila (Lee, 2003).

To further confirm the genetic interaction between tensin and the JNK pathway, the effect of tensin on JNK activity in vivo was tested. The extent of JNK phosphorylation was tested using anti-phosphospecific JNK antibody in the by overexpression line and the by² mutants. As expected, JNK phosphorylation is dramatically increased in the wing imaginal discs overexpressing by; this directly demonstrates increased JNK activity by by. On the contrary, JNK phosphorylation in the imaginal discs of the by² mutants is reduced compared with the control (Lee, 2003).

Moreover, the reduced size and the convex wing phenotype observed in the wings overexpressing by can be most easily explained by apoptosis in the wings. Since the induction of apoptosis by the JNK signaling is well established, it was expected that the wing phenotype induced by by overexpression might be due to apoptosis. To confirm the by-induced apoptosis in vivo, Acridine Orange staining of the relevant wing imaginal discs was carried out. As expected, the overexpression of by dramatically increases apoptotic cell death compared with the control (Lee, 2003).

Thus, tensin genetically interacts with the components of the JNK signaling pathway, and regulates JNK activity during wing development. The supporting evidence for the engagement of tensin in the JNK signaling pathway comes from a recent report that transfected mammalian tensin activates JNK signaling in HEK 293T cells (Katz, 2000). Interestingly, in mammalian cells, JNK is also activated via adaptor proteins p130 CAS and Crk which receive a signal from the FAK/Src tyrosine kinase complex in the cell adhesion sites when cells attach to the ECM. Since tensin is a possible substrate for FAK, and p130 CAS is able to interact with the C terminus of tensin, it is highly possible that tensin is involved in this signaling cascade and mediates signals from integrin and FAK to the JNK signaling pathway (Lee, 2003).

Immune activation of NF-kappaB and JNK requires Drosophila TAK1

Stimulation of the Drosophila immune response activates NF-kappaB and JNK signaling pathways. For example, infection by Gram-negative bacteria induces the Imd signaling pathway, leading to the activation of the NF-kappaB-like transcription factor Relish and the expression of a battery of genes encoding antimicrobial peptides. Bacterial infection also activates the JNK pathway, but the role of this pathway in the immune response has not yet been established. Genetic experiments suggest that the Drosophila homolog of the mammalian MAPK kinase kinase, TAK1 (transforming growth factor ß-activated kinase 1), activates both the JNK and NF-kappaB pathways following immune stimulation. Drosophila TAK1 functions as both the Drosophila IkappaB kinase-activating kinase and the JNK kinase-activating kinase. However, JNK signaling is not required for antimicrobial peptide gene expression but is required for the activation of other immune inducible genes, including Punch, Sulfated, and Malvolio (Mlv). Thus, JNK signaling appears to play an important role in the cellular immune response and the stress response (Silverman, 2003).

JNK signaling is also activated during the immune response in both flies and humans. However, the exact mechanism by which LPS leads to JNK activation in Drosophila is unclear, as is the role of JNK signaling during the immune response. Gene expression profiling has been used to infer that TAK1 is required for the activation of JNK signaling and that JNK signaling is important for wound healing. This study directly demonstrates that JNK activation requires TAK1. Thus, TAK1 appears to function both as a JNKK activating kinase and an IKK activating kinase, as proposed for mammalian TAK1. Furthermore, microarray results suggest that JNK signaling may have important functions in cellular immunity and the stress response (Silverman, 2003).

The gene expression profiling data presented in this study identifies a relatively small number of genes that specifically require the JNK signaling pathway for their LPS-induced expression. The expression of two genes (Punch and sulfated) identified in these experiments has been validated by real time RT-PCR. Punch is an immune inducible gene in cells in culture and in adult flies. However, although it has been shown that in adult flies the immune induction of Punch requires Relish (De Gregorio, 2001), the data presented in this study demonstrate that Punch induction in S2 cells requires JNK pathway components (hep, bsk, and TAK1) but not the Relish-activating kinase IKK. The experiments presented here were performed in an embryonic Drosophila cell line (that has macrophage-like qualities), whereas the data from De Gregorio (2001) was generated from entire adult flies. Thus, it is possible that the signaling pathways required for Punch induction vary depending on the developmental stage and cell type examined. In fact, Punch has at least two promoters that direct developmentally specific expression (Silverman, 2003).

Punch encodes the enzyme for GTP cyclohydrolase I, which is the first enzyme (and rate-determining step) in the formation of the cofactor tetrahydrobiopterin (BH4). This cofactor is required for the conversion of tyrosine to dopamine, which has at least two possible roles in immunity: (1) dopamine is one of two main Drosophila catecholamines, which are important for the stress response in both insects and mammals; (2) dopamine is the precursor of melanin, which is produced during wound healing and encapsulation processes in the fly. In fact, it has been proposed that increased Punch activity could lead to increased melanization (Silverman, 2003 and references therein).

The cofactor BH4 is also an essential cofactor for nitric oxide synthase (NOS). NO itself has at least two possible roles in the immune response: (1) NO is known to be a major microbicidal compound in mammalian phagocytic cells and is likely to function similarly in Drosophila macrophages; (2) NO has also been implicated in immune signaling in Drosophila. NO is required for transmitting a signal from the site of infection to the fat body, the major organ of immune responsive gene expression. Thus, Punch may contribute to the insect immune response in several ways, including protection against stress, melanization of wound sites, and activation of cellular and humoral immunity (Silverman, 2003 and references therein).

The potential role of sulfated in the immune response is less obvious. sulfated encodes an extracellular sulfatase that removes sulfate groups from heparin sulfate proteoglycans (HSPGs). In avian and Drosophila systems, it is thought that sulfated activity is crucial for the regulation of Wnt signaling, possibly by controlling the extracellular milieu in which the Wnt ligand travels (Silverman, 2003).

One of the most intriguing targets of both the JNK and IKK pathways is Mvl, the Drosophila NRAMP-1 homolog. Mvl mutants were first identified in the fly because they display gustatory behavioral defects caused by the inability to properly process sensory neuronal input. Mvl is expressed in both the nervous system and circulating hemocytes. In the mouse, NRAMP-1 is expressed in macrophages, and mutations in the NRAMP-1 gene are responsible for the sensitivity of some inbred mice strains to the Mycobacterium bovis bacille Calmette-Guérin (BCG) and other intracellular bacterial pathogens. NRAMP-1 is thought to control the levels of cations, possibly Fe²⁺ or Mn²⁺, in lysosomal compartments of mouse macrophages. A current model suggests that NRAMP-1 pumps cations out of the phagolysosome, thereby starving microbes of cations required by the enzymes (superoxide dismutase and catalase), which protect the bacteria from reactive oxygen intermediate (ROI)- and reactive nitrogen intermediate (RNI)-induced damage. In the fly, the role of Mvl in immunity is not yet characterized, but its induction during an immune response coupled with the activity of this protein in vertebrate macrophages suggests that it may play an important role in the cellular immune response (Silverman, 2003).

Microarray analysis has provided evidence that LPS-induced JNK activation is important for the stimulation of a gene expression program similar to that seen during dorsal closure. Thus, JNK may be important for wound healing. Expression of only a few of the JNK target genes (for example, Filamin) is reported in this study. The data argue that JNK signaling is required for the activation of cellular immunity and stress protection, whereas a connection to wound healing cannot be excluded by these data (Silverman, 2003).

Certain antimicrobial genes (e.g., diptericin) require a combination of transcription factors for their proper induction. It has been suggested, based on DNA footprinting and DNA sequence analysis, that Diptericin activation requires a kappaB binding site (now believed to be the site of Relish binding) as well as putative NF-IL6-like, and interferon regulatory factor (IRF)-like binding sites. However, of these only Relish is required for the immune inducible expression of diptericin. The data presented here show that the JNK signaling pathway and the AP-1-like factors activated by Drosophila JNK signaling are not involved in antimicrobial peptide gene induction in phagocytes. This would be quite different from immune activation of many mammalian cytokine genes, which require the coordination of several signaling pathways and the activity of several transcription factors for full immune induction. For example, IFN-ß induction requires the activation of three independent signaling cascades and the cooperative binding of three transcription factors, NF-kappaB, c-Jun/activating transcription factor 2 (ATF-2), and the interferon regulatory factor, to the enhancer region. Together, these transcription factors form a higher order complex known as the enhanceosome. Control of the insect antimicrobial genes may not require this complex enhancer architecture (Silverman, 2003).

These studies clearly demonstrate that activation of the innate immune response in Drosophila leads to the activation of JNK and NF-kappaB signaling pathways through a branched signal transduction cascade. The MAP3K TAK1 lies at the branch point of this cascade and likely functions as the JNKK activating kinase and the IKK activating kinase. These signaling pathways are highly conserved. TAK1 also serves similar functions in mammalian innate immune signaling. Furthermore, novel immune-induced targets of the JNK pathway have been identified, that may function in cellular immunity and stress protection (Silverman, 2003).

The TAK1 plays a pivotal role in the innate immune response of Drosophila by controlling the activation of JNK and NF-kappaB. Activation of TAK1 in mammals is mediated by two TAK1-binding proteins, TAB1 and TAB2, but the role of the TAB proteins in the immune response of Drosophila has not yet been established. A TAB2-like protein has been identified in Drosophila called dTAB2. Similar to mammalian TAB2/3, dTAB2 contains a ubiquitin-binding domain (N-CUE) at its N-terminal, and a highly conserved Zinc Finger (ZnF) domain at its C-terminal, respectively. dTAB2 can interact with TAK1, and stimulate the activation of the JNK and NF-kappaB signaling pathway. Furthermore, silencing of dTAB2 expression by dsRNAi inhibits JNK activation by peptidoglycans (PGN), but not by NaCl or sorbitol. In addition, suppression of dTAB2 blocks PGN-induced expression of antibacterial peptide genes, a function normally mediated by the activation of NF-kappaB signaling pathway. No significant effect on p38 activation by dTAB2 was found. These results suggest that dTAB2 is specifically required for PGN-induced activation of JNK and NF-kappaB signaling pathways (Zhuang, 2006).

Studies in mammalian system have shown that activation of TAK1 is regulated by two TAK1 binding proteins, TAB1 and TAB2/3. The dTAB2 appears to associate with dTAK1, thus dTAB2 also acts as a dTAK1-associating protein in Drosophila. In mammalian cells, TAB2 not only binds with TAK1, but also associates with an upstream regulator TRAF6 to form a TAK1-TAB2-TRAF6 complex. TRAF6 contains an N-terminal RING domain, and functions as a ubiquitin ligase, which, in conjunction with a dimeric Ub-conjugating enzyme complex consisting of Ubc13 and Uev1A.Mms2, to catalyze the K63-linked ubiquitination of TRAF6. TAB2 binds to K63-linked polyubiquitinated TRAF6 though a highly conserved C-terminal zinc finger (ZnF) domain, thus facilitating the activation of TAK1. In Drosophila, TAB2 also contains a conserved ZnF domain at its C-terminal, and deletion of this region eliminated its ability to activate the NF-kappaB signaling pathway. In addition, two TRAF homologues, dTRAF1 and dTRAF2, have been identified in Drosophila. Only dTRAF2 contains a RING domain, and dTRAF2 mutant has a reduced level of Diptericin and Drosomycin induction after E. coli infection. However, whether dTRAF2 can be polyubiquitinated and whether dTAB2 binds with polyubiquitinated dTRAF2 to facilitate the activation of dTAK1 still requires further biochemical study (Zhuang, 2006).

In mammals, the physiological importance of TAB2 has been clarified by studies on TAB2-deficient mice and RNAi studies. The phenotype generated from the TAB2 deficiency is very similar to that of NF-kappaB-, IKK-beta-, and NEMO/IKK-deficient mice. However, IL-1 or TNF-induced activation of NF-kappaB and JNK signaling pathways appears to be normal in TAB2-deficient embryonic fibroblasts. Recently, another mammalian TAB2 homologue, TAB3 has been identified, suppression of both TAB2 and TAB3 by RNAi inhibit the activation of IKK and JNK by IL-1 and TNF. In Drosophila, the generation of dTAB2-deficient fly has not been reported so far. However, silencing of dTAB2 by using dsRNAi inhibits PGN-induced activation of JNK and the expression of antibacterial peptide diptericin and attacin gene, two typical peptides produced when the IMD signaling pathway is activated by Gram-negative bacterial pathogens. Since the dTAB2 is associated with dTAK1 and TAB2 is believed to be a key regulator of TAK1, these results are consistent with previous studies that have shown that the Drosophila TAK1 is essential for the activation of both NF-kappaB and JNK MAP kinase signaling after PGN stimulation. Considering the association of dTAB2 with dTAK1 and the functional role of its mammalian homologue TAB2 in the activation of TAK1, the physiological function of dTAB2 in the stimulation of PGN is most likely mediated through TAK1 (Zhuang, 2006).

Compared to the studies of TAB2 and TAK1 in the activation of IKK and JNK, the functional role of TAB2 and TAK1 in the activation of p38 MAP kinase is less clear. TAK1 has been shown to directly phosphorylate and activate the MKK6, a MAP2K of p38 in vitro. However, no significant defect of PGN-induced p38 activation in TAB2-silenced Drosophila cells was observed. In addition, PGN-induced p38 activation is normal in TAK1-silenced cells. These results suggest that TAK1 and its associating protein TAB2 may not be involved in p38 activation in PGN stimulation. The activation of p38 by TAK1 in previous reports may have been caused by TAB1 protein, another component of TAK1 activation complex (Zhuang, 2006).

The Drosophila homolog of the putative phosphatidylserine receptor functions to inhibit apoptosis, perhaps acting through the JNK pathway

Exposure of phosphatidylserine is a conserved feature of apoptotic cells and is thought to act as a signal for engulfment of the cell corpse. A putative receptor for phosphatidylserine (PSR) was previously identified in mammalian systems. This receptor is proposed to function in engulfment of apoptotic cells, although gene ablation of PSR has resulted in a variety of phenotypes. The role of the predicted Drosophila homolog of PSR (dPSR) in apoptotic cell engulfment was examined and no obvious role for dPSR in apoptotic cell engulfment by phagocytes was found in the embryo. In addition, dPSR is localized to the nucleus, inconsistent with a role in apoptotic cell recognition. However, it was surprisind to find that overexpression of dPSR protects from apoptosis, while loss of dPSR enhances apoptosis in the developing eye. The increased apoptosis is mediated by the head involution defective (Wrinkled) gene product. In addition, the data suggest that dPSR acts through the c-Jun-NH2 terminal kinase pathway to alter the sensitivity to cell death (Krieser, 2007).

Evidence against a role for PSR in engulfment also comes from two other knockout models and from data on the localization of the protein. One of the reported mouse knockouts showed no difference in engulfment of apoptotic cells by macrophages in the mutant, although PSR^-/- macrophages were generally inhibited in their release of pro- and anti-inflammatory cytokines. In addition, fibroblast lines established from PSR^-/- embryos showed no defect in apoptotic cell engulfment or in their response to apoptotic cells. Zebrafish lacking PSR accumulated dead cells, but were not definitively shown to have defects in apoptotic cell engulfment. Finally, localization data from the current work and from a number of labs strongly supports a nuclear localization for the protein. This is not consistent with a role for PSR as a surface receptor for the recognition for apoptotic cells, although PS could theoretically modulate the activity of this protein within the cell (Krieser, 2007).

The observations support a role for dPSR in cell survival. In zebrafish, reduction of PSR resulted in an increase in the number of apoptotic cells present during development. In particular, the brains of these fish were shrunken and had an increase in apoptotic cells. In two of the mouse knockout models an increase in apoptotic cells was detected. However, all three knockouts resulted in perinatal lethality, with defects in differentiation in a variety of tissues. It is speculated that defects in engulfment detected in some of the gene ablation models could reflect a role for PSR in the proper differentiation of macrophages. Increased apoptosis seen in the current studies and by others might also be due to defects in proper differentiation in the absence of PSR (Krieser, 2007).

What insight can be gained from these studies into the function of dPSR in differentiation and cell survival? Increased dPSR results in a cell survival phenotype that is suppressed by activation of the JNK pathway, while loss of dPSR results in apoptosis, activated by the cell death regulator Hid, a known target of JNK activation in apoptosis. Taken together, these data suggest that dPSR may normally act to suppress JNK activation of Hid-induced apoptosis (Krieser, 2007).

JNK activation is important for many processes in cells, including cell death, proliferation and differentiation. A role for JNK in apoptosis was found in many mammalian cell types. Data from mouse knockouts of JNK also suggest a role for JNK in proliferation and differentiation. In addition, JNK activation in dying cells is required for proliferative signals originating from apoptotic cells in Drosophila. Interestingly, defects in proliferation and differentiation of many tissues were observed in mice that lack PSR. Taken together with observations of increased cell death in dPSR mutant flies, these observations suggest that some of the phenotypes seen in mouse and fish models of PSR gene ablation might be due to inappropriate activation of the JNK pathway (Krieser, 2007).

Based on genetic assays, it is proposed that one function of dPSR is to suppress Hid activation. Flies that lack dPSR show increased apoptosis in the developing pupal eye, which is suppressed in the absence of hid, while overexpression of dPSR results in ectopic cell survival. Hid function is required for the death of the interommatidial cells in the pupal retina. The results also showed that expression of dPSR can inhibit death induced by the expression of Hid- or Grim in the eye, and that loss of dPSR enhances Rpr-, Hid- or Grim-induced death in the eye. Interestingly, loss of one copy of hid can also suppress cell death induced by Rpr or Hid expression in the eye. Therefore alterations of dPSR levels in the eye may be altering Hid activity to modify the Grim- and Rprinduced eye phenotypes (Krieser, 2007).

JNK activation has been shown to increase hid activity. However, Hid activity is also modulated by activation of the Ras/Erk pathway. Ras activation results in the survival of ectopic interommatidial cells, through the downregulation of Hid activity. Ectopic Ras activation also results in genital rotation defects, similar to those seen with dPSR overexpression. This suggests that PSR overexpression might activate the Ras/Erk pathway. Based on the current data, it is not clear whether dPSR might activate Ras and thus suppress JNK activity, whether dPSR could suppress JNK and thus activate Ras, or whether dPSR might act independently in an opposing manner on the JNK and Ras pathways (Krieser, 2007).

By examining the function of dPSR in the Drosophila system, new insight has been provided into the controversy regarding this protein. Although no evidence was found that this protein plays a role in engulfment, it is important in cell survival. This is consistent with phenotypes seen in gene ablation models in other organisms. Furthermore, dPSR affects the JNK pathway, and this may provide a clue as to its diverse functions in mammals (Krieser, 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).

Proteins acting downstream of JNK

The activation of MAPKs is controlled by the balance between MAPK kinase and MAPK phosphatase activities. The latter is mediated by a subset of phosphatases with dual specificity (VH-1 family). This paper describes a new member of this family, encoded by the puckered gene of Drosophila. Mutations in this gene lead to cytoskeletal defects that result in a failure in dorsal closure 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. puckered expression is itself 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. 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. 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).

A model is proposed for the role of Puckered in dorsal closure in which signaling through Hep and Bsk leads to the expression of effectors of dorsal closure and a regulator encoded by puc. The function of the latter is to exert a negative feedback on the signaling cascade of hep and bsk. Interestingly, in mutants for Djun (a likely target of JNK activity), puc expression is absent at the leading edge of the epidermis (N. Perrimon, pers. comm. to Martin-Blanco, 1998), suggesting a transcriptional link between the activity of the JNK encoded by bsk and the expression of puc. Thus, the activation of MAPKs is controlled by the balance between MAPK kinase and MAPK phosphatase activities during dorsal closure. In this system, Puckered seems to act in a feedback loop. Puckered expression is upregulated by DJun and in turn, Puckered inactivates MAPK, whose function is the activation of DJun downstream of Rac signaling (Martin-Blanco, 1998).

What role is played by hemipterous and basket/DJNK in the process of dorsal closure? Specifically, what are the JNK phosphorylation targets in the process of 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 known to be involved in dorsal closure. Mutations in genes coding for several transcription factors produce dorsal open phenotypes, like pannier and serpent (two GATA transcript factors), and anterior open (an ETS domain protein also known as yan). 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 (Riego-Escovar, 1996).

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 of Dpp as such a ligand. Dorsal closure couples JNK and Dpp signaling pathways, a situation that may be conserved in vertebrate development (Glise, 1997).

The p150Spir protein provides a link between c-Jun N-terminal kinase function and actin reorganization

A direct link between JNK signaling and actin organization has not been found previously. The Jun N-terminal kinase (JNK, known as Basket in Drosophila) is a downstream effector of Rac and Cdc42 GTPases involved in actin reorganization. Basket plays a role in the regulation of cell shape changes and actin reorganization during the process of dorsal closure. One potential mechanism for induction of cytoskeletal changes by Basket is via transcriptional activation of the decapentaplegic gene, a member of the TGFbeta superfamily. Spire, termed p150-Spire in this study, has a docking site for Basket/JNK, at its carboxy-terminal end. In mouse fibroblasts, p150-Spir colocalizes with F-actin; its overexpression induces clustering of filamentous actin around the nucleus. When coexpressed with p150-Spire in NIH 3T3 cells, JNK translocates to and colocalizes with p150-Spire at discrete spots around the nucleus. Carboxy-termina l sequences of p150-Spir are phosphorylated by JNK both in vitro and in vivo. It is concluded that p150-Spir is a downstream target of JNK function and provides a direct link between JNK and actin organization (Otto, 2000).

p150Spire was identified in a yeast two-hybrid screen to search for novel Basket interaction partners. The cDNA encoding p150Spire contains an open reading frame of 1,020 amino acids. Transient transfection experiments in mouse fibroblasts reveal that the cDNA directs expression of a protein that migrates with an apparent molecular weight of approximately 150 kDa on an SDSñpolyacrylamide gel. The spire gene exhibits significant homology to a maternal gene pem-5 isolated from the ascidian Ciona savignyi. Comparison of Drosophila p150Spire sequences to human EST clones reveals a high conservation between the p150Spire sequences of Drosophila and human (Otto, 2000).

The p150Spire protein contains an acidic domain, a cluster of four WH2 domains, a modified FYVE zinc finger domain and a carboxy-terminal DEJL motif (docking site for Erk and JNK containing an LXL motif). WH2 domains bind monomeric actin: WH2 family proteins such as WASP and WAVE are involved in actin reorganization. Sequences encompassing the Spir WH2 domains interact directly with monomeric actin. As has been shown for WASP and WAVE, transient expression of p150Spire in adherent mammalian cells induces clusters of filamentous actin around the nucleus that colocalize with p150Spire in all transfected cells. A modified FYVE zinc-finger motif is located in the central region of the protein. The modified structure lacks a pocket of basic amino acids between cysteines 3 and 4 of the FYVE finger structures that is necessary for the binding of phosphatidylinositol 3-phosphate (Otto, 2000).

In this study, p150Spire was identified in a yeast two-hybrid screen as a DJNK-interacting protein. The smallest fragment that still binds DJNK consists of the carboxy-terminal 53 amino acids. A DEJL motif, characterized by a cluster of basic amino acids amino-terminal to an L/I-X-L/I motif (in the single-letter amino acid code), is located in this carboxy-terminal sequence (amino acids 997-1014 of p150Spire, KQKRSSARNRTIQNLTLD). DEJL domains have been shown to mediate the docking of the Erk and JNK mitogen-activated protein (MAP) kinases to either activating kinases or substrate proteins. To further characterize the interaction between p150Spire and JNK, the localization of the two proteins was determined when transiently expressed in NIH 3T3 cells. p150Spire accumulates in punctate spots located around the nucleus. Expression of a fusion protein between glutathione-S-transferase and JNK (GST-JNK) reveals both a cytoplasmic and nuclear distribution of the kinase. In the presence of p150Spire, GST-JNK is translocated from these areas and accumulates at places where p150Spire is located. The colocalization of the proteins is detected in all cells expressing both proteins but is not found in cells coexpressing GST alone with p150Spire (Otto, 2000).

Erk and JNK MAP kinases are recruited to substrate proteins via docking sites, enabling the kinases to phosphorylate serine or threonine residues adjacent to prolines (S/TP motifs). As p150Spire contains a JNK docking site (the DEJL motif) and several potential S/TP phosphorylation motifs, attempts were made to determine whether p150Spire is a phosphorylation target of JNK. A highly specific, constitutively active Erk can be generated by the fusion of Erk2 to its upstream activator Mek1 (Erk2-Mek1-LA). Similarly, JNK2 (rat) has been fused to its upstream activator MKK7 (mouse) via a linker region. The fusion protein, JNK-MKK7, is a constitutively active Jun N-terminal kinase. JNK-MKK7 phosphorylates amino-terminal c-Jun sequences in vitro and induces an electrophoretic mobility shift of the c-Jun protein when coexpressed in NIH 3T3 cells, indicating an in vivo c-Jun phosphorylation. MKK7 activates JNK by phosphorylating a TPY motif in the central region of JNK. JNK-MKK7 exhibits autophosphorylation and interacts with a phospho-specific antibody that recognizes activated JNK protein. A similar construct (JNKK2-JNK1) has been shown to be a specific, constitutively active c-Jun kinase (Otto, 2000).

An investigation was carried out to see whether p150Spire sequences are phosphorylated by JNK in vitro. Indeed, arsenite-activated JNK1, immunoprecipitated from NIH 3T3 cell lysates, phosphorylates a carboxy-terminal fragment of the p150Spire protein in an in vitro immune-complex kinase assay. Erk and p38 MAP kinases precipitated from the same lysates exhibit a basal phosphorylation activity that could not be increased by arsenite stimulation of the cells. Coexpression of p150Spire with MLK3-activated JNK2 [mixed lineage kinase (MLK3) is an upstream activator of JNKs] or JNK-MKK7 in mouse fibroblasts, induces several slower migrating forms of the p150Spire protein during SDS-PAGE (Otto, 2000).

The electrophoretic mobility shift of p150Spire strongly suggests that it is phosphorylated by JNK on multiple sites. The slower migrating forms are abolished by phosphatase treatment, demonstrating that the mobility shifts are due to phosphorylation of p150Spire protein. Supporting these data, a kinase-inactive mutant of JNK-MKK7 (containing the JNK mutations K55A and K56A and the MKK7 mutation K76E, called JNK-MKK7-KD) does not induce an electrophoretic mobility shift of p150Spire. To analyse the specificity of JNK phosphorylation of p150Spire in vivo, p150Spire was coexpressed with the constitutively active Erk2-Mek1-LA fusion protein. Although expressed to the same levels as JNK-MKK7, Erk2-Mek1-LA induces only a very weak electrophoretic mobility shift of p150Spire, indicating a preferential phosphorylation of p150Spire by JNK (Otto, 2000).

Drosophila Fos mediates ERK and JNK signals via distinct phosphorylation sites

During Drosophila development Fos acts downstream from the JNK pathway. It can also mediate ERK signaling in wing vein formation and photoreceptor differentiation. Drosophila JNK and ERK phosphorylate D-Fos with overlapping, but distinct, patterns. Analysis of flies expressing phosphorylation site point mutants of D-Fos reveals that the transcription factor responds differentially to JNK and ERK signals. Mutations in the phosphorylation sites for JNK interfere specifically with the biological effects of JNK activation, whereas mutations in ERK phosphorylation sites affect responses to the EGF receptor-Ras-ERK pathway. These results indicate that the distinction between ERK and JNK signals can be made at the level of D-Fos, and that different pathway-specific phosphorylated forms of the protein can elicit different responses (Ciapponi, 2001).

The loss of wing vein tissue on expression of D-Fos^bZIP resembles phenotypes resulting from defects in the Drosophila epidermal growth factor receptor (DER) pathway, caused, for example, by loss-of-function alleles of DER itself or of other genes required for DER signaling, such as rhomboid, vein, ras, and ERK/rolled. Therefore, the above results might indicate that D-Fos acts as a mediator of the DER/ERK signaling pathway during wing vein differentiation. The artificial activation of this RTK pathway, by gain-of-function alleles or by overexpression of downstream effectors, gives rise to ectopic veins in the wing. To establish whether D-Fos might act epistatically to DER, and whether D-Fos^bZIP or a reduced D-fos gene dose (kayak alleles) might suppress such a phenotype was examined (Ciapponi, 2001).

The Ellipse^B1 (Elp^B1) allele of DER represents an activated component of the DER/ERK signaling pathway. In addition to other phenotypes, for example, in the eye, Elp^B1 animals consistently develop wings with ectopic wing vein material. Strikingly, both D-Fos^bZIP expression in the wing imaginal disc (using the 32B Gal4 driver) or the removal of one copy of D-fos in a kay heterozygote, suppresses this phenotype almost completely. To confirm that the observed effect is specific and caused by a reduction of endogenous D-Fos function, add-back experiments were performed in which this reduction was compensated by supplying extra wild-type D-Fos from a transgene, driven by the heat shock promoter (hs D-fos. Significantly, the presence of the D-fos transgene abrogates the suppression of Elp^B1 by kay and reinstates the extra vein phenotype caused by elevated DER activity. This result confirms that the suppression of the activated DER allele is due to a loss of D-fos activity. Hence, D-Fos mediates wing vein patterning downstream from or in parallel with DER (Ciapponi, 2001).

Next, whether D-Fos mediates ERK signaling also during eye morphogenesis was investigated. Defects in photoreceptor differentiation can be induced by the RTK gain-of-function alleles Elp^B1 and sev^S11. The Elp^B1 allele dominantly causes an abnormal eye phenotype that manifests itself in roughness and the occasional lack of outer photoreceptors. This phenotype can be suppressed largely by the removal of one copy of D-fos and restored subsequently by simultaneous transgenic expression of wild-type D-Fos. A gain-of-function transgene of the RTK-coding gene sevenless (sev^S11) causes the characteristic appearance of ectopic R7 photoreceptor cells in nearly all ommatidia. The sev^S11 phenotype can be suppressed by the expression of dominant-negative Fos. In flies carrying sev^S11 in a heterozygous kay² background, the ectopic R7 photoreceptor phenotype is suppressed significantly; the number of normal ommatidia increases from 5% to approximately 20%. Reintroduction of D-fos by a transgene in this double mutant background restores the percentage of ommatidia with extra photoreceptors observed in sev^S11 heterozygous animals. Taken together, these results indicate that D-Fos can act as a rate-limiting component downstream from the RTKs Sev and DER during eye development (Ciapponi, 2001).

Considering that D-Fos is a transcription factor and based on the precedents of D-Jun and c-Fos, the most obvious role for D-Fos in DER and Sev signal transduction would be that of an effector of the Drosophila MAP kinase Rolled. Therefore, the effect of reducing D-fos activity in animals expressing the gain-of-function allele rl^Sem was examined. Expression of Rl^Sem under UAS control in the wing imaginal disc results in an extra-vein phenotype, markedly when the flies are reared at 25°C and milder at 18°C. Simultaneous expression of D-Fos^bZIP along with Rl^Sem causes a striking suppression of ectopic vein formation, whereas additional expression of full-length D-Fos leads to a strong enhancement of the phenotype. To confirm that the observed suppression of the rl^Sem phenotype was not due to an effect of D-Fos on the transgene promoters, a similar genetic interaction experiment was performed using the endogenous rl^Sem gain-of-function allele and kay² allele. kay² heterozygosity suppresses the rl^Sem-induced extra-vein phenotype. This effect is reverted by ubiquitous expression of D-Fos, indicating that the suppression is due specifically to the decreased activity of D-Fos. These genetic interactions confirm that the role of D-Fos in RTK signal is that of an effector of Rolled (Ciapponi, 2001).

The results described above reveal D-Fos as a downstream component of the ERK signal transduction pathway, yet previous genetic analyses have shown that the transcription factor serves as an effector of JNK. This raises the question of whether the function of D-Fos as recipient of ERK or JNK is mutually exclusive and determined by the cellular context, or whether the transcription factor may mediate both JNK and ERK responses in one tissue or one cell. The developing eye provides a system to approach such a question. Biochemical and genetic studies have indicated that the planar polarity pathway downstream from Frizzled (Fz) and Dishevelled (Dsh) leads to the activation of a JNK-type MAPK module. During retinal morphogenesis, this pathway controls the mirror-symmetric arrangement of ommatidial units relative to the dorso-ventral midline. Thus, in the developing eye the activity of JNK and ERK signal transduction can be monitored separately in vivo (by planar polarity and R-cell recruitment, respectively) (Ciapponi, 2001).

To determine whether D-Fos is involved in planar polarity signaling, the effect of D-Fos^bZIP expressed under the control of Gal4 drivers in the developing eye was examined. When D-Fos function is thus reduced, a striking combined phenocopy of defects in ERK and JNK signal transduction ensues. Sections of eyes of the hairy Gal4/UAS D-fos^bZIP or of the sev Gal4/UAS D-fos^bZIP genotypes display both a lack of photoreceptor cells, diagnostic of inadequate ERK signal transduction, and misoriented ommatidia, indicating defects in planar polarity signaling. This mutant phenotype makes it plausible that D-Fos, in addition to its role in photoreceptor cell recruitment downstream from ERK, acts as an effector of JNK signaling in planar polarity determination. Therefore, D-Fos mediates both JNK and ERK responses in a defined group of cells of the developing retina (Ciapponi, 2001).

To investigate the potential regulation of D-Fos by protein phosphorylation, in vitro kinase assays were performed in which recombinant JNK/Bsk and ERK/Rl were used as kinases and different bacterially expressed versions of D-Fos were used as substrates. In this in vitro setting, both Bsk and Rl could phosphorylate full-length D-Fos. To get an initial indication as to which residues might serve as target sites for ERK or/and JNK, the D-Fos amino acid sequence was compared with that of mammalian Jun and Fos proteins. The sequence alignments identified several sequences in D-Fos with similarity to confirmed JNK or ERK phosphorylation sites in the mammalian molecules. T89 and T93 of D-Fos correspond in their sequence context and relative location to established JNK phosphorylation sites in c-Jun. Alignment of the C-terminal parts of D-Fos and c-Fos show a conserved residue (T584) that corresponds to a previously described MAPK phosphorylation site in c-Fos. Moreover, several serine or threonine residues were identified that might serve as target sites for the proline-directed MAPKs. Mutant derivatives of D-Fos were generated in which one or more of these candidate phosphorylation sites was substituted by alanine. JNK/Bsk, but not ERK/Rl, can efficiently phosphorylate a fragment spanning the 170 N-terminal amino acids of D-Fos. When alanine substitutions are introduced in positions T89 and T93, the N-terminal D-Fos fragment is no longer an efficient substrate for JNK phosphorylation (Ciapponi, 2001).

Additional N-terminal residues that conform to the S/TP consensus (T234, S235, T237, and T254) are phosphorylated by neither JNK nor ERK. Similar to the 170-amino-acid N-terminal fragment, a D-Fos fragment covering the N-terminal 285 amino acids is not a substrate for ERK. This indicates that the N-terminal part of D-Fos is a good substrate for JNK/Bsk but not for ERK/Rl, and that the residues T89 and T93 serve as the main N-terminal JNK target sites (Ciapponi, 2001).

Interestingly, a small deletion that removes a sequence with remote similarity to the c-Jun delta-domain (amino acids 28-56), but that does not span the phosphorylation sites T89 and T93, completely abrogates phosphorylation of the D-Fos N-terminal fragment by JNK/Bsk. It is possible that this deletion destroys a delta-domain-like JNK docking site present in D-Fos (Ciapponi, 2001).

Next, the C-terminal part of D-Fos was analyzed; it contains seven potential phosphorylation sites for MAPKs. A fragment spanning the C-terminal 280 amino acids of D-Fos is, in contrast to the N terminus, phosphorylated efficiently by both Bsk and Rl. Alanine substitutions in all seven putative MAPKs target sites causes complete loss of phosphorylation. However, mutating subsets of the seven putative phosphorylation sites does not result in a complete loss of phosphorylation by either Rl or Bsk, indicating the presence of multiple phosphorylation sites in the C-terminal part of D-Fos. These results indicate that, at least in vitro, D-Fos is a direct substrate of both Drosophila JNK and ERK and that it contains overlapping, but distinct, sets of phosphorylation sites for the two kinases (Ciapponi, 2001).

After establishing which residues serve as substrates for JNK and/or ERK in vitro, it was important to determine the regulatory relevance of these sites in vivo. Transgenic fly strains expressing mutant forms of full-length D-Fos were generated in which either the putative N-terminal, JNK-specific phosphorylation sites, or the C-terminally located ERK and JNK substrate sites were replaced by alanine (D-Fos^N-Ala and D-Fos^C-Ala. In the D-Fos^{pan Ala} mutant, all the putative MAPK phosphorylation sites, both N- and C-terminal, were substituted by alanine. When the nonphosphorylatable D-Fos^{pan Ala} was expressed from a UAS-driven transgene under the control of the epidermal driver 69B Gal4, it gave rise to a strong thoracic cleft at the dorsal midline, resembling kay or hep mutants, or phenotypes that result from D-Fos^bZIP expression. Evidently, D-Fos^{pan Ala} represents a dominant-negative mutant that can interfere with JNK-dependent thorax closure (Ciapponi, 2001).

The relevance was investigated of subgroups of the D-Fos MAPK phosphorylation sites during thorax closure. Expression of D-Fos^N-Ala results in a distinctive thorax cleft, although not quite as pronounced as in the case of D-Fos^{pan Ala}. Importantly, this result shows that the N-terminal, JNK-specific phsosphorylation sites in D-Fos are required for a well-defined JNK-dependent developmental mechanism. Expression of D-Fos^C-Ala, which lacks the C-terminal phosphorylation sites, or of D-Fos^wt has no discernible effect. These results indicate that either the C-terminal sites play only an ancillary role and are not essential for JNK signaling in the signal transduction pathway controlling thorax, or that this mutant does not compete well with endogenous D-Fos and therefore has no dominant-negative effect in this context (Ciapponi, 2001).

Next, whether expression of the different phosphorylation mutants of D-Fos might also interfere with ERK responses was investigated. Expression of D-Fos^{pan Ala} in the posterior compartment of wing imaginal disc (using en Gal4) causes loss of wing vein material typical of mutants defective in DER to Rolled signaling. Thus, consistent with the observation that D-Fos^{pan Ala} has lost all substrate sites for both Rolled and Bsk, it acts as a dominant-negative form that interferes with D-Fos function in both the ERK and the JNK pathways (Ciapponi, 2001).

Interestingly, however, the D-Fos^N-Ala and D-Fos^C-Ala mutants influence the ERK and JNK response differently. D-Fos^N-Ala, which interfers dominantly with JNK-mediated thorax closure, has no effect on ERK-dependent wing vein formation. This is consistent with these sites not being substrates for Rolled. The D-Fos^C-Ala mutant, however, which is neutral in thorax development, causes loss-of-vein phenotype (Ciapponi, 2001).

To examine whether differential phosphorylation of D-Fos might also be used in the developing eye to distinguish between ERK and JNK signaling, the effect of the D-Fos^Ala mutants on ERK-dependent photoreceptor cell recruitment and JNK-mediated ommatidial rotation was examined. Different D-Fos^Ala mutants were expressed along with sev^S11 in the eye imaginal disc under the control of the sevenless enhancer. As in the case of wing vein formation, D-Fos^N-Ala does not alter the sev^S11 phenotype, whereas the expression of D-Fos^C-Ala or of D-Fos^{pan Ala} causes a significant suppression of the extra R7-cell recruitment. Thus, ERK signaling, whether it is triggered by DER or by Sev, appears to require only the C-terminal phosphorylation sites of D-Fos (Ciapponi, 2001).

Next, whether the D-Fos^Ala mutants could suppress the ommatidial misrotation phenotype that is elicited by overexpression of Fz and the ensuing activation of JNK was tested. Coexpression of all Ala mutants and Fz under the control of sev Gal4 leads to a significant suppression of the misrotation phenotype observed in flies expressing Fz alone. Wild-type D-Fos does not have this effect. These results indicate that the JNK-phosphorylation sites of D-Fos are required for the manifestation of the Fz gain-of-function phenotype. The Fz-JNK response in the eye is also affected by mutation of the C-terminal sites that do not visibly disturb the thorax closure response. This might be explained by the higher sensitivity of the ommatidial rotation paradigm or a higher relative expression of the Fos^Ala mutant transgene in the photoreceptor cells (Ciapponi, 2001).

The dominant phenotypic effects of D-Fos^Ala expression support the interpretation that Fos is regulated by protein phosphorylation to mediate developmental decisions and indicates that the residues identified by mutagenesis and in vitro kinase assay are required for in vivo function. Moreover, these findings indicate clearly that the function of D-Fos as a mediator of JNK/Bsk and ERK/Rl cascades is in both cases that of a direct kinase substrate (Ciapponi, 2001).

An important question raised by the finding that D-Fos can mediate signaling by both ERK and JNK is how the decision between these distinct cellular responses is made, that is, how the cell 'knows' which program to execute when D-Fos becomes phosphorylated. Several mechanisms have been suggested to contribute to signal specificity in such situations, in which one protein mediates different cellular responses. One model proposes a combinatorial mechanism by which several factors with overlapping broad responsiveness have to cooperate to mediate a defined specific cellular behavior. However, the observation that D-Fos participates in ERK as well as JNK signal transduction in the same group of cells of the developing Drosophila eye, by regulating photoreceptor differentiation and ommatidial rotation, respectively, argues against cell type-specific cofactors that modulate the response to D-Fos phosphorylation. The distinct substrate sites in D-Fos phosphorylated by ERK and JNK raise a novel possibility to explain the signal-specific D-Fos response. It is suggested that D-Fos exists in two different activated forms, depending on whether it is phosphorylated by ERK or JNK. These differentially phosphorylated forms might then selectively trigger either the ERK or the JNK response. This idea is supported by in vivo experiments in which phosphorylation site-specific point mutants of D-Fos were expressed in the developing fly. A mutant that lacks all phosphorylation sites interferes dominantly with both ERK and JNK signaling in thorax closure, the wing, and the eye imaginal disc, supporting further the general relevance of D-Fos phosphorylation in developmental decisions. D-Fos mutants lacking subsets of phosphorylation sites, however, affected JNK and ERK signal responses differentially. An N-terminal cluster of JNK sites that is not phosphorylated by ERK is critical for the JNK response in vivo. A mutant lacking these sites interfers with thorax closure and planar polarity regulations, both bona fide JNK responses, but not with wing vein formation or photoreceptor differentiation, which are regulated by ERK. Conversely, a mutant that removes all ERK substrate sites dominantly suppresses processes normally controlled by this MAP kinase. These data indicate that signal-responsive transcription factors, such as D-Fos, may have different signal-specific functions. It is tempting to speculate that such a mechanism might be used by other signaling proteins that are receptive to different upstream signals (Ciapponi, 2001).

Eyes absent mediates cross-talk between retinal determination genes and the receptor tyrosine kinase signaling pathway: A possible role for the p38 MAPKs in activation of Eya

Drosophila Eyes absent function is positively regulated by mitogen-activated protein kinase (MAPK)-mediated phosphorylation: this regulation extends to developmental contexts independent of eye determination. In vivo genetic analyses, together with in vitro kinase assay results, demonstrate that Eya is a substrate for extracellular signal-regulated kinase, the MAPK acting downstream in the receptor tyrosine kinase (RTK) signaling pathway. Thus, phosphorylation of Eya appears to provide a direct regulatory link between the RTK/Ras/MAPK signaling cascade and the retinal determination gene network (Hsiao, 2001).

To address the possibility that Eya might be a direct downstream target of the RTK pathway, the Eya sequence was examined for potential MAPK phosphorylation consensus sites, defined as P-X-S/T-P (P, proline; X, any amino acid; S/T, serine or threonine). Investigations of alternative possibilities, including direct transcriptional regulation or protein-protein interactions between Yan and Eya, have yielded negative results to date. Two sites matching the consensus were found and will be referred to as 'MAPK sites.' Both MAPK sites are located ~80 residues upstream of the highly conserved 'Eya domain,' which has been shown to mediate interactions with So and Dac proteins. Examination of mammalian Eya protein sequences reveals similarly located MAPK sites in mouse and human Eya1, Eya2, and Eya4, suggesting that MAPK phosphorylation might be important for an evolutionarily conserved aspect of Eya function or regulation. Eya1 contains two MAPK sites, whereas Eya2 and Eya4 retain the second site but have a less stringent two amino acid consensus, S/T-P, at the first. No consensus MAPK sites were found in Eya3, although the less stringent two amino acid consensus is present at the second site (Hsiao, 2001).

To investigate the importance of the two MAPK phosphorylation sites with respect to Drosophila Eya function, the effects of mutating the phosphoacceptor residues were assessed in transgenic flies using an ectopic eye induction assay. First, site-directed mutagenesis was used to replace the serine residues in both sites with alanine (referred to as Eya^S-A), effectively destroying the MAPK sites. Second, the serine residues were mutated to glutamic or aspartic acid (referred to as Eya^S-D/E) to mimic the negative charge associated with phosphorylation. Transgenic lines expressing wild-type Eya (referred to as Eya^WT), Eya^S-A, and Eya^S-D/E under control of the UAS promoter were used to induce tissue-specific expression. For each construct, ten independent lines were assayed, and the data from the strongest and weakest were discarded. For the remaining eight lines, ~300 progeny of the appropriate genotype were scored for each cross, and the percentage of flies exhibiting ectopic eye induction was calculated (Hsiao, 2001).

On average, expression of Eya^WT induced ectopic eye formation in ~50% of the flies assayed. Most ectopic eyes were found on the head near the eye and around the base of the antenna. Patches of red eye pigment were also frequently induced on the wing hinges and underside of the metathoracic legs as well as occasionally on the wings, legs, and antennae. As expected when surveying a collection of independent transgenic insertions, positional effects resulted in a fairly broad distribution of phenotypic strengths. Thus, penetrance of ectopic eye induction ranged from almost 80% in the strongest line to <5% in the weakest. Strikingly, when the nonphosphorylatable Eya^S-A transgene is expressed, the average incidence of ectopic eye formation dropped to ~20% as compared with the 50% observed with Eya^WT. In addition, the size of the ectopic eye patches is noticeably reduced in Eya^S-A. Thus, on average, mutation of the two MAPK sites to a nonphosphorylatable form reduces Eya's effectiveness at inducing ectopic eye formation (Hsiao, 2001).

In the converse experiment in which the MAPK sites were mutated so as to mimic the constitutively phosphorylated state (Eya^S-D/E), the incidence of ectopic eye formation increased to an average value of ~80%. Furthermore, flies expressing Eya^S-D/E exhibit an increase in the size and number of ectopic eye patches relative to flies expressing Eya^WT. For example, in Eya^WT lines, ectopic eyes usually form on only one side of the head, whereas in Eya^S-D/E lines, ectopic eye induction often occurs bilaterally. Thus, mimicking the constitutively phosphorylated state dramatically enhances the efficacy of the Eya transgene in promoting ectopic eye formation. As with Eya^S-A, Eya^S-D/E lines exhibited no obvious changes in expression, stability, or subcellular distribution of the protein product that might provide alternate explanations for the apparent increase in activity (Hsiao, 2001).

To assess whether both MAPK sites are critical for Eya function, transgenes containing single MAPK site mutations were assayed for ability to promote ectopic eye formation. These transgenes are referred to as Eya^1S-A, Eya^2S-A, Eya^1S-D, and Eya^2S-E, where 1 and 2 refer to the specific MAPK site mutated. Expression of these transgenes produces intermediate results compared with the lines in which both MAPK sites were altered. For example, both Eya^1S-A and Eya^2S-A transgenes exhibited an average of ~30% ectopic eye induction, a value that represents an increase relative to Eya^S-A but a decrease relative to Eya^WT. Similarly, both Eya^1S-D and Eya^2S-E transgenes exhibited an intermediate value of ~65%, suggesting reduced activity relative to Eya^S-D/E but increased activity relative to Eya^WT. Together, these results suggest that both MAPK sites additively contribute to Eya function and regulation (Hsiao, 2001).

When interpreting the consequences of overexpressing a transgene, genetic determination of whether the observed phenotype reflects an antimorphic (dominant negative) or a hypermorphic (dominant activated) effect helps in assessing whether the particular transgene is functioning in a positive or negative manner relative to other regulators of the pathway. By genetically reducing the dose of the endogenous gene, it is possible to distinguish between the two possibilities (Hsiao, 2001).

To confirm that the ectopic eye induction associated with expression of the Eya transgenes reflects an increase in Eya activity, it was asked whether a 50% reduction in dosage of endogenous eya could suppress the phenotype. In all three cases (Eya^WT, Eya^S-A, and Eya^S-D/E), flies heterozygous for a null eya allele showsweaker and less penetrant phenotypes. For both Eya^WT and Eya^S-A, a 50% reduction in ectopic eye formation is observed. Suppression is less pronounced with Eya^S-D/E transgenes, consistent with the interpretation that Eya^S-D/E is a hyperactivated protein (Hsiao, 2001).

These results, in conjunction with the finding that the Eya^S-A and Eya^S-D/E products are less and more efficient at promoting ectopic eye induction, respectively, suggest that Eya function is positively regulated via phosphorylation of these two MAPK sites in vivo. The fact that some activity is retained in the Eya^S-A transgenes implies that MAPK phosphorylation does not provide a simple on/off switch, but rather serves to modulate the level of Eya activity (Hsiao, 2001).

The results obtained from overexpressing the Eya^WT, Eya^S-A, and Eya^S-D/E transgenes implicate MAPK phosphorylation as positively regulating Eya activity. Drosophila, like mammals, has multiple MAPK family members, including ERK, that mediates RTK/Ras initiated signals, the Jun N-terminal Kinase (JNK), and the p38 stress-responsive MAPKs. Less is known about the upstream signaling mechanisms that activate JNK and p38 MAPKs, although both families respond to stress and may exhibit some functional redundancy. Although isolation of Eya in an RTK pathway-based genetic screen made ERK the best candidate, other MAPK family members could also potentially phosphorylate Eya. Currently, specific loss-of-function mutations are available for only two members of the MAPK family in Drosophila: ERK and JNK. The gene rolled encodes ERK, whereas the gene basket encodes JNK. Two stress-responsive MAPKs, p38a and p38b, have been cloned and characterized in overexpression assays, although specific mutations have not yet been reported (Hsiao, 2001).

To address whether phosphorylation of Eya by MAPK is physiologically relevant, it was asked whether genetically reducing the dose of either ERK (rolled) or JNK (basket) could suppress the phenotypes associated with Eya overexpression. rolled mutations dramatically suppresses Eya^WT phenotypes, whereas basket mutations have no effect. Strong suppression of Eya^WT phenotypes was also obtained with mutations in the Epidermal growth factor receptor (Egfr), suggesting that activity of the canonical RTK/Ras/MAPK cascade participates in modulating Eya activity. Together, these results suggest that in vivo, ERK, the MAPK responsive to RTK-initiated signals, but not JNK, positively regulates Eya activity. A double mutant between rolled and basket suppresses Eya^WT at the same level as rolled alone, suggesting there are no synergistic effects between these two kinases. In a related experiment, it was found that coexpressing Eya^WT and an activated allele of ERK enhances the penetrance and severity of the phenotypes, whereas coexpression of Eya^WT and JNK do not (Hsiao, 2001).

Interestingly, a reduction in rolled dosage is also able to suppress the phenotypes associated with Eya^S-A transgenes. Because the two MAPK sites are effectively destroyed in this construct, the suppression is unlikely to reflect a direct interaction between ERK and the Eya^S-A product. Two possible interpretations could explain this result: (1) additional sites in Eya could be phosphorylated by ERK, apart from the two MAPK consensus sites identified; (2) the suppression could simply reflect downregulation of endogenous Eya. The fact that reduction in rolled dosage suppresses to a greater extent than reduction in eya dosage is consistent with this interpretation, assuming that the pool of endogenous ERK is limiting. In this case, then the amount of ERK produced in a heterozygous rolled background would be insufficient to activate even 50% of the endogenous Eya, and thus, even stronger suppression would be obtained relative to that seen with a 50% reduction in endogenous eya (Hsiao, 2001).

Consideration of the genetic interaction data obtained with the Eya^S-D/E transgenes increases the likelihood of the second scenario. A 2-fold reduction of either endogenous eya or endogenous rolled led to a comparable ~15% reduction in penetrance of ectopic eye induction in the Eya^S-D/E background. If Eya function were regulated via MAPK phosphorylation at additional sites, reducing the dose of rolled should have a greater impact on the phenotype than reducing the dose of eya, even in the hyper-activated Eya^S-D/E background. Thus, the explanation is favored that suppression of Eya^S-A and Eya^S-D/E upon reduction of endogenous ERK is primarily attributable to the dose sensitivity of the phenotype relative to endogenous Eya activity. The fact that suppression of Eya^S-D/E is less striking than that observed in either the Eya^WT or the Eya^S-A backgrounds is consistent with the hypermorphic nature of the Eya^S-D/E product (Hsiao, 2001).

To complement the in vivo genetic experiments and to determine whether the interaction between Eya and MAPK is direct, kinase assays were performed to assess which MAPK phosphorylates Eya preferentially in vitro. Activated ERK or JNK were immunoprecipitated from Drosophila S2 cultured cells transfected with appropriate cDNA constructs. Both kinases were active, as demonstrated by their ability to phosphorylate myelin basic protein and GST-Yan. GST-Eya^WT and GST-Eya^S-A fusion proteins were generated and tested for the ability to serve as ERK or JNK substrates. Consistent with the in vivo genetic results, ERK phosphorylates GST-Eya^WT, whereas JNK, although clearly active, does not (Hsiao, 2001).

The p38a and p38b MAPKs were also tested for ability to phosphorylate Eya using the in vitro kinase assay. Both p38a and p38b phosphorylated GST-Eya^WT at levels comparable to those obtained with ERK. These results suggest a possible role for the p38 MAPKs in activation of Eya, although in vivo validation of these predictions must await the isolation of specific mutations in p38a and p38b. However, given the recent report suggesting that p38b MAPK functions downstream of dpp signaling, it is tempting to speculate that the kinase assay results may provide a mechanistic basis underlying the genetic synergy that has been demonstrated between dpp and the retinal determination genes (Hsiao, 2001).

Over the course of the experiments looking at frequency of ectopic eye induction, several phenotypes associated with Eya overexpression were noticed that had not been previously reported. Specifically, Eya overexpression results in wing defects, increased number of scutellar macrochaetae, and problems with thoracic closure. Additional phenotypes, including rough eyes and arista to leg transformations, were observed at low penetrance and have not been extensively characterized (Hsiao, 2001).

Although eya loss-of-function phenotypes have not been reported in adult tissues other than the eye and germ line, it was found that all of the phenotypes associated with Eya overexpression can be suppressed by a 50% reduction in endogenous eya. This argues strongly that eya is expressed and could play a role in the normal development of these tissues. Furthermore, similarly strong suppression is achieved upon reducing the dose of endogenous dac or so, suggesting that part or all of retinal determination gene network may be redeployed in developmental contexts independent of eye formation (Hsiao, 2001).

Genetic tests to determine which MAPK might be responsible for modulating Eya activity in these contexts were performed as described for the ectopic eye phenotype. Because the thoracic closure and macrochaetae phenotypes were more variable and therefore less conducive to quantitative analyses, focus was placed on Eya regulation in the wing. A reduction in rolled dosage strongly suppresses whereas a reduction in basket dosage has little effect on the observed wing phenotypes. Conversely, coexpression of Eya^WT and activated ERK enhances the severity of the wing defects, whereas coexpression of JNK has little effect. Because expression of activated ERK alone results in ectopic wing vein formation, it is formally possible that the enhancement is additive rather than synergistic. However, within the same wing, the two phenotypes are easily distinguished, and the best interpretation is that the Eya-associated defects are enhanced whereas the ERK-associated ones are not. As was found in the ectopic eye formation assay, mutations in Egfr also suppressed the wing phenotypes. Thus, in the contexts of both eye and wing development, Eya activity appears to be modulated by ERK, the MAPK functioning downstream of the RTK signaling pathway (Hsiao, 2001).

These results suggest RTK signaling plays dual and opposing roles during eye development. Previous work has indicated that an early, MAPK-independent aspect of RTK signaling antagonizes the decision to become an eye. The current study shows that a MAPK-dependent signal positively promotes eye morphogenesis later in development by directly modulating Eya activity. The fact that the same pathway can exert such apparently opposing effects is not unusual; depending on the specific context, the same signal may be interpreted in strikingly different ways (Hsiao, 2001 and references therein).

JNK signaling pathway is required for efficient wound healing in Drosophila

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 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).

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).

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 DFos^bZip 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 gen