basket/JNK
Early DJNK staining is detected in cleavage stage embryos and appears to represent expression of maternal mRNA. Zygotic DJNK staining is present in all germ layers during germ-band extension. During germ-band retraction, the same intense staining remains in the epidermis and the central nervous system. When dorsal closure begins, Basket/JNK staining is increased in the CNS and is present in the lateral epidermal region extending up to the amnioserosa. At stage 15, DJNK stains in the ventral nerve cord, the brain and some peripheral neurons (Sluss, 1996).
In head midline structures, in particular the optic lobe and stomatogastric nervous system, there may be a late phase of EGFR signaling (as assayed by the expression of aos and activated ERK) whose significance is not yet known. EGFR signaling could be involved in modifying the inhibitory feed-back loop between neurogenic and proneural genes that exists in other neurectoderm cells. In the head midline neurectoderm, regulation of proneural and neurogenic genes has to be different. Thus, instead of a short burst of proneural gene expression in proneural clusters that is resolved into expression in individual neuroblasts, proneural genes are expressed for a long period of time; at the same time, the expression is never restricted to single neuroblasts. Since genes of the E(spl) complex are expressed in the same cells that express lsc, the inhibitory loop between E(spl)-C and proneural genes must be interrupted at some level. It is possible that Egfr signaling is causing the interruption of this inhibitory loop. Based on genetic studies of Notch and Egfr signaling in the compound eye, it has been speculated that one of the consequences of Egfr activation (which ultimately is required for all ommatidial cell types to differentiate) is to inhibit N signaling, since constitutively active N inhibits ommatidial cell differentiation by preventing response to differentiative signals. However, the same effect could be achieved if Egfr signaling, similar to what is proposed here for the midline neurectoderm, interrupts the inhibition of proneural genes by E(spl). Although this would not prevent N signaling, it would cancel the effect of N signaling on downregulating proneural genes and thereby keep cells in a state of competency to respond to signals (Dumstrei, 1998).
During dorsal closure in Drosophila melanogaster, cells of the lateral epidermis migrate over the amnioserosa to encase the embryo. At least three classes of dorsal-open group gene products are necessary for this morphogenetic movement. Class I genes code for structural proteins that effect changes in epidermal cell shape and motility, including zipper, coracle, canoe and myospheroid. Class II and III genes code for regulatory components of closure: Class II genes encode Drosophila Jun amino (N)-terminal kinase (DJNK) signaling molecules, including misshapen, hemipterous, basket, Jun-related antigen, kayak, anterior open/yan and puckered, and Class III genes encode Decapentaplegic-mediated signaling molecules. All characterized dorsal-open group gene products function in the epidermis. Reported here is a molecular and genetic characterization of raw, a newly defined member of the Class II dorsal-open group genes. The novel protein encoded by raw is required for restriction of DJNK signaling to leading edge epidermal cells as well as for proper development of the amnioserosa. Taken together, these results demonstrate a role for Raw in restriction of epidermal signaling during closure and suggest that this effect may be mediated via the amnioserosa (Byars, 1999).
The distribution of raw transcripts, which is uniform in early embryos, becomes more refined as development proceeds. In gastrulating embryos, raw transcripts are evident in the midgut rudiments as well as in the ventral furrow and cephalic and dorsal folds. In germband-extended-stage embryos, raw transcripts appear limited to the gut primordia and ventral neurogenic region. This spatial restriction continues in germband-retracted-stage embryos where transcription is most conspicuous in the central nervous system (CNS) and midgut. Although raw is transcribed throughout the CNS in later stages, its pattern of expression is refined in the midgut. Here, raw transcription is limited primarily to the second midgut constriction. The patterns of raw transcription observed correlate well with documented roles for raw in the nervous system and intestinal tract; however, they provide little insight into how Raw might affect dorsal closure. All previously characterized dorsal-open group genes are expressed in the epidermis or in the epidermis and amnioserosa, and raw could not be detected in either of these tissues. Reports that amnioserosal gene transcription is sometimes recalcitrant to staining in standard in situ staining protocols led the authors to evaluate raw expression by other means. More notably, reporter expression allowed for detection of beta-galactosidase in the amnioserosa (Byars, 1999).
Raw function could affect dorsal closure at any of several steps in the morphogenetic pathway. It could play a structural role in either the amnioserosa or the epidermis. As examples, Raw might be required for the physical interaction of the opposing tissues during closure or for the restructuring of the cytoskeleton that is associated with changes in epidermal cell shape during closure. Alternatively, Raw could play a signaling role, providing either an amnioserosal or epidermal cue that regulates signaling in the epidermis during closure. In an effort to distinguish between these structural and instructive models and to understand better the role of Raw in dorsal closure, epidermal morphology and signaling was examined Epidermal cell elongation, a hallmark of dorsal closure, is thought to be driven by the movement of a myosin motor with a filamentous actin (F-actin) substrate in leading edge cells. Several lines of evidence indicate that this initiating event of closure occurs normally in raw mutant embryos. Proper F-actin localization has been documented in raw mutant embryos by staining with phalloidin. The fact that F-actin accumulates normally in leading edge epidermal cells and that these cells can elongate in animals harboring either hypomorphic or amorphic raw alleles indicates that dorsal closure initiates properly in raw mutants. Moreover, these data suggest that the DJNK signaling pathway is operative in the epidermal leading edge of raw mutant embryos (Byars, 1999).
To more directly test DJNK activation in raw mutants, expression of dpp and puc, the two known transcriptionally regulated targets of DJNK signaling during closure, were examined. In wild-type embryos, epidermal expression of both dpp and puc is dependent upon DJNK signaling and is confined to leading edge cells. Transcription of these targets is abolished in leading edge epidermal cells in hep (DJNKK), bsk (DJNK) and Jra (DJun) mutant embryos, and expanded in embryos overexpressing either activated c-Jun or wild-type DJun. As seen in embryos with ectopic DJun function, the domains of dpp and puc transcription in the epidermis of raw mutant embryos are markedly expanded beyond their normal ranges. It was also noted that puc transcription, as assayed by an enhancer reporter, expands to a greater lateral distance in raw mutants than in puc mutants. These data demonstrate that Raw is required for restriction of dpp and puc to the leading edge of the dorsal epidermis, and point to an upstream role for Raw in negatively modulating DJNK signaling during closure (Byars, 1999).
To establish a regulatory link between raw and Jra, their epistatic relationship was determined. Embryos doubly mutant for raw and Jra were scored for the appearance of alternative dpp expression phenotypes (either missing from leading edge epidermal cells, as in Jra mutants or ectopic epidermal expression, as in raw mutants). The finding that dpp is not expressed in leading edge epidermal cells in raw;Jra double mutants defines Jra as epistatic to raw and confirms the hypothesis that raw functions upstream of the DJNK signaling pathway (Byars, 1999).
In summary, distinct features of functionality define raw as unique. The raw gene is the first of the dorsal-open group for which defects in gene expression have been documented in the amnioserosa. More notably, raw represents the first of the dorsal-open group mutants to show gross defects in dorsal closure that are attributable to a gain in DJNK signaling rather than a loss of DJNK signaling. The characterization of raw as a novel upstream component of the dorsal closure pathway represents an important first step in understanding the mechanism of regulating DJNK signaling during closure (Byars, 1999).
Additional clues as to the function of raw come from a second analysis of the role of raw in dorsal closure. Mutations in two genes, rib and raw cause defects in the morphology of a number of tissues in homozygous mutant embryos. A variety of tubular epithelial tissues adopt a wide, round shape in mutants and dorsal closure fails. Cells of the normal tubular epithelia are columnar and wedge-shaped, and cells of the epidermis become elongated dorsoventrally as dorsal closure occurs. However, the cells of mutants are round or cuboidal in all of the tissues with mutant phenotypes, consistent with the hypothesis that the products of these genes are required for proper cell shape. Cytoskeletal defects, in particular, defects in myosin-driven contraction of the cortical actin cytoskeleton, could be responsible for the lack of specific cell shapes in mutant embryos. This possibility is supported by the observation that the intracellular localization of nonmuscle myosin to the leading edge of the dorsally closing epidermis is absent or reduced in rib and raw mutant embryos. In contrast, the band of actin that is also located at the leading edge is neither eliminated nor interrupted by either rib or raw mutations. Furthermore, mutations of zipper, the gene encoding the nonmuscle myosin heavy chain, exhibit mutant phenotypes in most of the same tissues affected by rib and raw, and many of the phenotypes are similar to those of rib and raw. Therefore, the products of rib and raw may be required for proper myosin-driven contraction of the actin cytoskeleton (Blake, 1998).
Various lines of evidence from mammalian tissue culture suggest that Cdc42 functions in regulating the JNK signaling cascade. In Drosophila, the JNK pathway plays an integral role in dorsal closure, a morphogenetic process involving cell shape changes and local signaling events that occurs late in embryogenesis. One demonstrated function of the JNK pathway is to promote expression of the morphogen Decapentaplegic in cells at the leading edge of the lateral epidermis during dorsal closure. Consistent with this notion, previous studies have shown that dpp expression in the leading-edge epidermal cells is disrupted in embryos carrying mutations in members of the JNK signaling pathway. Reduction but not complete loss of maternally contributed hemipterous results in complete loss of dpp expression while loss of a negative regulator, puckered, results in increased dpp expression (Genova, 2000 and references therein).
Thus dpp expression at the leading edge is sensitive to the level of JNK pathway function. Previous studies using ectopic expression of dominant Cdc42 alleles have suggested that Cdc42 is necessary for dorsal closure and functions upstream of the JNK pathway at the leading edge. If this is so, then loss of Cdc42 function should disrupt JNK signaling and therefore dpp expression in these cells. To test this hypothesis, in situ hybridization to dpp mRNA was performed on embryos derived from Cdc424/Cdc426 mothers. Although ~70% of embryos produced by these females displayed epithelial defects and lethality, normal levels of dpp expression were observed in all embryos that developed to the onset of dorsal closure, including those that had arrested development due to insufficient levels of Cdc42 function. Thus, unlike known upstream components of the JNK pathway, reduction in Cdc42 function has no apparent effect on dpp expression by leading-edge cells at the time of dorsal closure (Genova, 2000).
Shark (SH2 domain ankyrin repeat kinase) is a Drosophila nonreceptor tyrosine kinase that contains from amino to carboxyl terminus, a Src homology 2 (SH2) domain (N-SH2), five ankyrin repeats, a second SH2 domain (C-SH2), a proline-rich and basic region, and a tyrosine kinase domain. Analysis of the phenotypes associated with a shark loss-of-function mutation demonstrate that Shark activity is essential for the migration of the dorsolateral epidermis of the embryo during dorsal closure (DC). Shark kinase functions in DC upstream of Dpp expression by leading edge (LE) cells (Fernandez, 2000).
Because no obvious genetic interactions were obtained between Shark and mutations of the JNK pathway, tests were performed to see whether constitutive activation of the JNK or the Dpp pathway could rescue the shark1 DC phenotype. When shark1 GLCs were generated in the background of flies carrying a shark1 chromosome with an inserted transposon expressing an activated form of c-Jun (hs-SEjunAsp), shark1 DC defects were completely rescued, in some cases, as determined by the decreased penetrance of embryonic lethality (~10% lower than the fully penetrant 50% observed without the expression of hs-SEjunAsp) and by the complete or partial enclosure observed in unhatched embryos. These results are consistent with the action of Shark upstream of bsk (JNK) in the JNK pathway in LE cells (Fernandez, 2000).
Cancer is a multistep process involving cooperation between oncogenic or tumor suppressor mutations and interactions between the tumor and surrounding normal tissue. This study is the first description of cooperative tumorigenesis in Drosophila, and uses a system that mimics the development of tumors in mammals. The MARCM system was used to generate mutant clones of the apical-basal cell polarity tumor suppressor gene, scribbled, in the context of normal tissue. scribbled mutant clones in the eye disc exhibit ectopic expression of cyclin E and ectopic cell cycles, but do not overgrow due to increased cell death mediated by the JNK pathway and the surrounding wild-type tissue. In contrast, when oncogenic Ras or Notch is expressed within the scribbled mutant clones, cell death is prevented and neoplastic tumors develop. This demonstrates that, in Drosophila, activated alleles of Ras and Notch can act as cooperating oncogenes in the development of epithelial tumors, and highlights the importance of epithelial polarity regulators in restraining oncogenes and preventing tumor formation (Brumby, 2003).
A clonal approach, more closely resembling the clonal nature of mammalian cancer, was used to analyze the effects of removing Scrib function on tumor formation. This analysis indicates that Drosophila scrib- tumors: (1) lose tissue architecture, including apical-basal cell polarity; (2) fail to differentiate properly; (3) exert non-cell-autonomous effects upon the surrounding wild-type tissue; (4) upregulate cyclin E and undergo excessive cell proliferation; (5) are restrained from overgrowing by the surrounding wild-type tissue via a JNK-dependent apoptotic response, and (6) show strong cooperation with oncogenic alleles of Ras and Notch to produce large amorphous tumors. These conclusions are summarized in a model for tumor development in Drosophila. It is suggested that the role of epithelial cell polarity regulators in restraining oncogenes is likely to be of general significance in mammalian tumorigenesis (Brumby, 2003).
The model suggests that the wild-type larval eye disc is a monolayered columnar epithelium, in which cell proliferation is tightly regulated. Cell architecture is maintained by the formation of adherens junctions, the apical localization of Scribbled, and adhesion to the basement membrane. Mutation of scrib results in loss of apical-basal polarity, leading to multilayering and rounding up of cells. scrib- tissue also shows impaired differentiation, and ectopic cyclin E expression (by an unknown mechanism) leads to ectopic cell proliferation. Unrestrained overgrowth and tumor formation of scrib- cells is held in check by compensatory JNK-mediated apoptosis, dependent upon the presence of surrounding wild-type cells. Secondary mutations are required to avoid this apoptotic fate. If JNK activity is blocked within scrib- cells, by expressing a dominant-negative form of JNK, apoptosis is prevented, resulting in tissue overgrowth and lethality. Even more aggressive overgrowth results from the addition of activating oncogenic alleles of Ras or Notch. In addition to promoting cell survival, these oncogenes must also promote tumor cell proliferation; however, it is proposed that other downstream effectors of these oncogenes are likely also to be important, since it was not possible to mimic the cooperative overgrowth effects of RasACT or NACT on scrib- tissue by simply blocking apoptosis and enhancing cell proliferation (Brumby, 2003).
Overproliferation of scrib- clones in the eye disc is compensated for by JNK-mediated apoptosis. Blocking JNK pathway activity in scrib- eye clones greatly increases the proportion of clonal tissue, and results in lethality to the host. Since downregulating JNK pathway activity in otherwise wild-type clones of tissue does not induce increased cell proliferation, it is suggested that JNK pathway activity in scrib- clones induces apoptosis. This is consistent with previous reports on the pro-apoptotic effects of the JNK pathway in the Drosophila eye and the current observations. Recent studies in mammals would also suggest that activation of the JNK pathway can limit the growth of tumors in situ, possibly by increasing apoptosis (Brumby, 2003).
How JNK-mediated apoptosis is induced in scrib- clones is not known. While Scrib could play a direct role in repressing JNK pathway activity, it is also possible that the JNK pathway is activated indirectly, in response to other cellular defects. In the wing disc, removal of cells by JNK-mediated apoptosis is linked to discontinuities in a cell's response to morphogen gradients, most notably the antero-posterior patterning regulator, Dpp, in a process probably related to cell competition, with the purpose of eliminating aberrant or slow growing cells. Although this form of compensatory JNK-mediated apoptosis has not yet been demonstrated within the eye disc, the observation that the surrounding wild-type tissue context plays an important role in limiting the overgrowth of scrib- tissue argues against a simple cell-intrinsic apoptotic response of scrib- cells to a loss of cell polarity, and is more consistent with an integrative response mediated by both the tumor cells and the surrounding wild-type cells, as exemplified by cell competition. Whether this is dependent on a failure of scrib- cells to transduce Dpp signaling is not known; however, other interesting possibilities also warrant further investigation. Notable is the recent identification of a tumor necrosis factor-induced apoptotic signaling pathway involving the JNK pathway. It is also important to keep in mind the involvement of the JNK pathway in orchestrating cell shape changes during the morphogenetic movements of dorsal and thorax closure and wound healing. clones of scrib- tissue expressing BskDN (JNKDN) appear morphologically different from those expressing the apoptosis inhibitor p35; most notably, the clones are generally larger and less rounded than those expressing p35. This would imply either that p35 is not as effective as BskDN in preventing cell death, or that there are other effectors of the JNK pathway that are important in the inhibition of scrib- tumor overgrowth. The possibility that JNK activation could play a role in eliminating scrib- tissue from the epithelium in a process reminiscent of wound healing is currently being investigated (Brumby, 2003).
dlg and lgl mutant clones also show poor viability, suggesting that JNK-mediated apoptosis could be a common response to the loss of cell polarity and overproliferation induced by the absence of these tumor suppressors. Indeed, while other regulators of epithelial cell polarity, such as Crumbs and E-cadherin, apparently do not act as tumor suppressors in Drosophila, the effects of these mutations on cell proliferation when cell death is blocked warrant further examination. Interestingly, in mammalian systems, the polarized nature of epithelia is also important in protecting cells from an apoptotic response, and this acts as a brake on tumor development when polarity is disrupted (Brumby, 2003).
The tumor suppressor retinoblastoma protein (pRb) is inactivated in a wide variety of cancers. While its role during cell cycle is well characterized, little is known about its properties on apoptosis regulation and apoptosis-induced cell responses. pRb shorter forms that can modulate pRB apoptotic properties, resulting from cleavages at caspase specific sites are observed in several cellular contexts. A bioinformatics analysis showed that a putative caspase cleavage site (TELD) is found in the Drosophila homologue of pRb (RBF) at a position similar to the site generating the p76Rb form in mammals. Thus, this study generated a punctual mutant form of RBF in which the aspartate of the TELD site is replaced by an alanine. This mutant form, RBFD253A, conserved the JNK-dependent pro-apoptotic properties of RBF but gained the ability of inducing overgrowth phenotypes in adult wings. This overgrowth is a consequence of an abnormal proliferation in wing imaginal discs, which depends on the JNK pathway activation but not on wingless (wg) ectopic expression. These results show for the first time that the TELD site of RBF could be important to control the function of RBF in tissue homeostasis in vivo (Milet, 2014. PubMed).
Mutations that inhibit differentiation in stem cell lineages are a common early step in cancer development, but precisely how a loss of differentiation initiates tumorigenesis is unclear. This study investigated Drosophila intestinal stem cell (ISC) tumours generated by suppressing Notch(N) signalling, which blocks differentiation. Notch-defective ISCs require stress-induced divisions for tumour initiation and an autocrine EGFR ligand, Spitz, during early tumour growth. On achieving a critical mass these tumours displace surrounding enterocytes, competing with them for basement membrane space and causing their detachment, extrusion and apoptosis. This loss of epithelial integrity induces JNK and Yki/YAP activity in enterocytes and, consequently, their expression of stress-dependent cytokines (Upd2, Upd3). These paracrine signals, normally used within the stem cell niche to trigger regeneration, propel tumour growth without the need for secondary mutations in growth signalling pathways. The appropriation of niche signalling by differentiation-defective stem cells may be a common mechanism of early tumorigenesis (Patel, 2015).
This paper described a step-wise series of events during the earliest stage of tumour development in a stem cell niche. First, the combination of environmentally triggered mitogenic signalling and a mutation that compromises differentiation generates small clusters of differentiation-defective stem-like cells. Autocrine (Spi/EGFR) signalling between these cells then promotes their expansion into clusters, which quickly reach a size capable of physically disrupting the surrounding epithelium and driving the detachment and apical extrusion of surrounding epithelial cells (that is, ECs). This loss of normal cells seems to involve tumour cell/epithelial cell competition through integrin-mediated adhesion. Subsequently, the loss of epithelial integrity (specifically, EC detachment) triggers stress signalling (JNK, Yki/YAP) in the surrounding epithelium and underlying VM, and these stressed tissues respond by producing cytokines (Upd2,3) and growth factors (Vn, Pvf, Wg, dILP3). These signals are normally used within the niche to activate stem cells for epithelial repair, but in this context they further stimulate tumour growth in a positive feedback loop. It is noteworthy that in this example a single mutation that blocks differentiation is sufficient to drive early tumour development, even without secondary mutations in growth signalling pathways that might make the tumour-initiating cells growth factor- and niche-independent (for example, Ras, PTEN). Thus, tumour cell-niche interactions can be sufficient to allow tumour-initiating cells to rapidly expand, increasing their chance to acquire secondary mutations that might enhance their growth or allow them to survive outside their normal niche. This study highlights the importance of investigating the factors that control paracrine stem cell mitogens and survival signals in the niche environment. Tumour-niche interactions may be important to acquire a sizable tumour mass before the recruitment of a tumour-specific microenvironment that supports further tumour progression. A careful analysis of similar interactions in other epithelia, such as in the lung, skin or intestine could yield insights relevant to the early detection, treatment and prevention of cancers in such tissues (Patel, 2015).
Injury is an inevitable part of life, making wound healing essential for survival. In postembryonic skin, wound closure requires that epidermal cells recognize the presence of a gap and change their behavior to migrate across it. In Drosophila larvae, wound closure requires two signaling pathways [the Jun N-terminal kinase (JNK) pathway and the Pvr receptor tyrosine kinase signaling pathway] and regulation of the actin cytoskeleton. In this and other systems, it remains unclear how the signaling pathways that initiate wound closure connect to the actin regulators that help execute wound-induced cell migrations. This study shows that chickadee, which encodes the Drosophila Profilin, a protein important for actin filament recycling and cell migration during development, is required for the physiological process of larval epidermal wound closure. After injury, chickadee is transcriptionally upregulated in cells proximal to the wound. JNK, but not Pvr, mediates the increase in chic transcription through the Jun and Fos transcription factors. Finally, it was shown that chic-deficient larvae fail to form a robust actin cable along the wound edge and also fail to form normal filopodial and lamellipodial extensions into the wound gap. These results thus connect a factor that regulates actin monomer recycling to the JNK signaling pathway during wound closure. They also reveal a physiological function for an important developmental regulator of actin and begin to tease out the logic of how the wound repair response is organized (Brock, 2012).
The traditional model of the actin cytoskeleton in cell migration, based on in vitro cell culture and biochemical assays, provides a useful framework for the mechanics of how cell migration is regulated. However, there is need for in vivo studies in order to answer important questions that are not addressed by the current model: 1. Is there a role for Profilin-mediated recycling during wound-induced migration of differentiated cells in vivo? 2. Is there a role for transcriptional regulation of actin regulators during such migrations? This latter question emerges because the basic model generally assumes that migratory cells have an intact actin-regulatory apparatus that needs only to be activated to initiate and sustain migration. While this assumption may be correct for migrating cells in developmental contexts one could imagine that initially non-migratory differentiated cells may need more than their resting complement of actin regulators in order to effect long-distance migration (Brock, 2012).
Unwounded larval epidermal cells have an even distribution of actin and Profilin throughout the cytoplasm and are thought to be non-migratory. These fully differentiated epithelial cells secrete an apical cuticle and a basal lamina. They respond to the physiological signal of tissue damage by partially dedifferentiating and becoming migratory. This study shows that the leading edge cells form multiple actin-based structures including a discontinuous cable, filopodia, and lamellipodia. A working model is proposed where the basal levels of Profilin are sufficient to make actin-based structures, but wound-induced transcription of chic is required for the cells to efficiently migrate. The lack of actin-based structures at the wound edge in cells lacking Profilin would indicate that if Formin-mediated actin nucleation is involved in wound healing, it likely requires Profilin. An epidermal sheet lacking detectable Profilin fails to close wounds or form any actin-based structures at the wound edge whereas a sheet containing only a basal level of Profilin (i.e., one that is lacking proteins that transcriptionally regulate Profilin after wounding, such as JNK, Fos, or Jun) forms actin structures at the wound edge, but is ultimately unable to efficiently migrate and close the wound. This model is complicated by the fact that cells lacking JNK, Fos, or Jun also have defects in dedifferentiation, as these cells do not stop secreting cuticle following wounding. Thus, the possibility cannot completely excluded that the lack of wound closure is due to defects in dedifferentiation. However, it is entirely possible that upregulation of actin-binding regulators is an important component of the dedifferentiation process, as this involves returning to a state during which these cells were competent to migrate (Brock, 2012).
Current wound closure models have identified two signaling pathways that are important for healing. One is Pvr signaling, where the secreted VEGF-like ligand Pvf1 activates the Pvr receptor. Currently, only a few proteins are suspected of being downstream of Pvr signaling, but Profilin is not among them. Given that epidermal cells lacking Pvr are unable to mobilize actin to the wound edge, Pvr is likely upstream of actin regulatory proteins that initiate actin polymerization at the leading edge of migrating cells. The second pathway is JNK signaling, which is required for closure but not for actin polymerization at the wound edge. Naively, it was anticipated that wound-induced chic expression would be regulated by Pvr since epidermal expression of UAS-chicRNAi also blocks actin accumulation at the wound edge. Surprisingly, this is not the case. chiclacZ expression is instead regulated by JNK signaling, as it is in the developing embryo during DC . This data reveals that although the JNK signaling pathway is not required for actin nucleation at the wound edge it contributes to actin dynamics through regulating expression of chic and perhaps other genes important for migration (Brock, 2012).
How does JNK signaling activate chic transcription after wounding? Although the upstream signal for the JNK signaling pathway is still unknown, the kinase cascade is well-defined and is thought to culminate with the phosphorylation of the transcription factors, DJun and DFos. These two proteins are commonly thought to act as a dimer (AP-1) to mediate transcriptional activation of target genes. In the early DC studies chickadee expression was shown to depend on the JNK signaling pathway. This study did not address the roles of DJun and DFos in particular, although these transcription factors are required for DC. In wound healing contexts, however, it appears that DFos can act without DJun to activate a ddc-wound reporter and a msn-lacZ wound reporter. This study found that both DJun and DFos are required to activate chic. Additionally, two consensus binding sequences for the AP-1 transcription factor (TGANTCA) are located upstream of the chic start codon (depending on the message isoform the sites are located in the 5’UTR, the first intron, or the promoter region), indicating that it is at least possible that the upregulation of chic transcription is directly accomplished by Jun and Fos. The consensus sequence is also located upstream of the human Pfn1, indicating that there is potential for this regulation to be conserved. This suggests that in the migrating cells at the wound edge, DFos can act either as a homodimer, with unidentified binding partners, or with DJun to regulate the necessary transcriptional targets (Brock, 2012).
In Drosophila embryonic models of wound closure both the contractile actin cable and filopodial processes are important for wound closure, but their relative contributions are still unclear. There has been debate over whether the cable mediates closure through contraction, through serving as a platform for extension of processes into the wound gap, or through a combinaton of these functions. From the data shown in this study it seems that actin-based contraction is not a major contributor to larval wound closure. First, the actin concentrations that appear at larval wound edges are discontinuous. Second they do not appear to be locally contractile given that the cells behind prominent concentrations do not obviously taper toward the wound. This is similar to what has been observed in the embryonic Xenopus epithelium where actin cables form but differently shaped wounds do not round up as would be expected from cable contraction. Thus it would appear that in larvae the actin concentrated at the wound edge primarily facilitates process extension into the wound gap (Brock, 2012).
This study has establish a connection between a known wound-induced signaling pathway, JNK signaling, and Profilin-mediated regulation of the actin cytoskeleton. It is speculated that transcriptional induction of actin-regulators may be a general feature of cell migration in differentiated cells as suggested by a recent study of cells undergoing EMT. By connecting upstream signaling pathways to downstream actin dynamics, this work begins to unravel the logic of how the cellular movements required for wound closure are orchestrated (Brock, 2012).
Apoptotic cells can produce signals to instruct cells in their local environment, including ones that stimulate engulfment and proliferation. This study identified a novel mode of communication by which apoptotic cells induce additional apoptosis in the same tissue. Strong induction of apoptosis in one compartment of the Drosophila wing disc causes apoptosis of cells in the other compartment, indicating that dying cells can release long-range death factors. Eiger, the Drosophila tumor necrosis factor (TNF) homolog, was identified as the signal responsible for apoptosis-induced apoptosis (AiA). Eiger is produced in apoptotic cells and, through activation of the c-Jun N-terminal kinase (JNK) pathway, is able to propagate the initial apoptotic stimulus. During coordinated cell death of hair follicle cells in mice, TNF-alpha is expressed in apoptotic cells and is required for normal cell death. AiA provides a mechanism to explain cohort behavior of dying cells that is seen both in normal development and under pathological conditions (Perez-Garijo, 2013)
It is becoming clear that apoptosis is not a passive phenomenon where dying cells merely die and are silently removed from the tissues. Instead, apoptotic cells have the capacity to produce proliferative signals, such as Wg and Dpp, thus serving as a crucial driving force in wound healing, regeneration and tumor formation in a variety of different organisms. This study demonstrates that signaling by apoptotic cells is not limited to the production of proliferative signals: dying cells can generate pro-apoptotic signals that induce apoptosis in neighboring cells. The induction of this apoptosis is triggered by Eiger, the ortholog of TNF in Drosophila, which in turn activates the JNK pathway and leads to cell death in a non-autonomous manner. Furthermore, evidence is provided for the existence of AiA under physiological conditions in mice. During catagen, the regressive phase of the hair cycle, apoptotic cells in the lower and transient portion of the HF also express TNF-α. Significantly, TNF-α is required for coordinated cell death in the HF. Taken together, these results suggest that AiA plays an important physiological role for the coordination of cohort cell death (Perez-Garijo, 2013)
These experiments demonstrate that induction of apoptosis in one compartment results in induction of non-autonomous apoptosis in the neighboring compartment. This is true under many different conditions: both when undead cells (expressing rpr/hid and baculovirus caspase inhibitor p35) were generated or upon induction of genuine apoptosis (expressing rpr/hid alone); once there is ectopic expression of mitogens that leads to excessive proliferation and growth or while blocking mitogenic production or growth of the compartment (Perez-Garijo, 2013)
One intriguing observation is that this non-autonomous cell death usually displays a pattern consisting of two groups of cells in the wing pouch. One possible explanation for this is that the affected cells are the most susceptible to the death signal. In fact, the regions of the wing pouch where the non-autonomous cell death is observed are also more prone to cell death as a response to different apoptotic stimuli, such as irradiation or hid over-expression (Perez-Garijo, 2013)
Another possibility to explain why cell death is observed at a distance would be that dying cells are producing other signals that inhibit apoptosis. This protective signal would diffuse only short range, and in this way the distance of cells to the border would determine the ratio between the pro-apoptotic and the protective signal, tipping the balance in favor of death or survival. In fact, it has been shown that cells neighboring apoptotic cells downregulate Hippo pathway and consequently activate Diap1. Another good candidate for such an anti-apoptotic signal would be Wg, as it is expressed in an opposite pattern from the non-autonomous apoptosis and is also diffusing from apoptotic cells in the posterior compartment. However, this study attempted to modify Wg levels in different ways and no changes were observed in the apoptosis pattern (Perez-Garijo, 2013)
In contrast, in physiological conditions such as the coordinated cell death of hair follicle (HF) cells observed in mice, it would be expected that signaling between apoptotic cells would occur at a much shorter range, probably affecting the immediate neighbors. In any case, the observation that TNF-α is exclusively detected in apoptotic cells and the fact that its inhibition leads to desynchronization of the HF cycle strongly suggests that AiA can be a mechanism to coordinate cell death within a tissue (Perez-Garijo, 2013)
In these experimental systems, AiA requires both the TNF and JNK signaling pathways. Eiger is produced by apoptotic cells in the posterior compartment of the wing disc and it activates JNK in cells of the neighboring compartment, inducing them to die. Downregulation of Eiger in the posterior compartment or JNK in the anterior compartment is able to suppress AiA. However, it remains to be elucidated whether Eiger directly diffuses to the cells in the anterior compartment, or if some other mechanism is responsible for the activation of JNK in dying cells in the anterior compartment. Recently, it was shown that, upon wounding, JNK activity can be propagated at a distance through a feed-forward loop (Wu, 2010). Significantly, AiA is not restricted to the Drosophila wing disc. Evidence was obtained for a role of TNF-α-mediated AiA during the destruction of the hair follicle (HF) in catagen, the regressive phase of the hair cycle. TNF-α plays a known role to promote cell death and has been previously implicated in HF progression, wound healing and regeneration. However, the cellular source of TNF-α remained unknown and it was previously not appreciated that apoptotic cells can be the source of these signals. These results suggest that AiA and at least some of the underlying mechanism have been conserved in evolution to promote coordinated cell death (Perez-Garijo, 2013)
The observation that apoptotic cells can signal to other cells in their environment and instruct them to die has potentially many important implications. On the one hand, there are situations where propagation of an apoptotic stimulus may be a useful mechanism to achieve the rapid and coordinated death of large cell populations. The experiments in mice show that this can be the case during the catagen phase of the HF cycle. There are many other examples of cell death being used during development to sculpt tissues and organs, including the removal of structures during metamorphosis (tadpole tail, larval organs in insects, elimination of inappropriate sex organs in mammals, deletion of the amnioserosa during insect embryogenesis) and the separation of digits through apoptosis of the interdigital webbing in many vertebrates. In all these cases, AiA may facilitate cohort behavior and contribute to the rapid and complete elimination of large fields of cells (Perez-Garijo, 2013)
Propagation of cell death may also be an efficient way to prevent infection. It is known that cells respond to viral infection by entering apoptosis and in this way impede the replication of the virus. The process of AiA would extend apoptosis to the neighboring cells, preventing also their infection and thus avoiding the spread of the virus (Perez-Garijo, 2013)
However, propagation of apoptosis may be detrimental in pathological conditions where excessive cell death underlies the etiology of the disease. This may be the case for neurodegenerative disorders, hepatic diseases, cardiac infarction, etc. In all these cases it remains to be studied whether extensive amounts of apoptosis that are observed in the affected tissues are a direct consequence of cell damage in an autonomous manner or if part of the cell loss could be attributed to a process of propagation through AiA (Perez-Garijo, 2013)
Finally, AiA may play a role in cancer. It is known that radiotherapy in humans can induce biological effects in non-irradiated cells at a considerable distance, a phenomenon called radiation-induced bystander effect. The current findings provide a possible explanation for some of these effects. Therefore, large-scale induction of apoptosis by AiA may contribute to successful cancer therapy. TNF family proteins are being used as models for drug development aimed to treat cancer). Furthermore, Eiger, the only TNF member in Drosophila, has a known role in the elimination of pre-tumoral scrib- clones. In addition, cell competition induces cell death even in aggressive scrib-RasV12 tumors, raising the possibility that AiA is induced during tumor initiation, which may affect the tumor microenvironment and ultimately tumor growth. It is well known that TNF can play both tumor-promoting and tumor-suppressing roles, but AiA has not been investigated in this context. Future studies will shed new light on the relevance of signaling by apoptotic cells and the implications of this signaling mechanism in different scenarios (Perez-Garijo, 2013)
Genetic analyses in Drosophila revealed a synergy between Notch and the pleiotropic transcription factor Mef2 (myocyte enhancer factor 2), which profoundly influences proliferation and metastasis. This study shows that these hyperproliferative and invasive Drosophila phenotypes are attributed to upregulation of eiger, a member of the tumour necrosis factor superfamily of ligands, and the consequent activation of Jun N-terminal kinase signalling, which in turn triggers the expression of the invasive marker MMP1. Expression studies in human breast tumour samples demonstrate correlation between Notch and Mef2 paralogues and support the notion that Notch-MEF2 synergy may be significant for modulating human mammary oncogenesis (Pallavi, 2012).
A genetic modifier screen was undertaken in Drosophila and a number of genetic modifiers of Notch signals were identified that affect proliferation. Further examination of one of these modifiers, Mef2, established that its synergy with Notch signals directly triggers expression of the Drosophila JNK pathway ligand eiger, consequently activating JNK signalling that profoundly influences proliferation and metastatic behaviour. It might perhaps be worth noting that metastatic behaviour in Drosophila may not be completely equivalent to mammalian metastasis, notwithstanding the fact that they share molecular signatures, for example, MMP activation (Pallavi, 2012).
Cancer is characterized by the deregulation of the balance between differentiation, proliferation and apoptosis; thus, it is not surprising that the Notch signalling pathway, which plays a central role in all these developmental events, is increasingly implicated in oncogenic events. The rationale of this study is based on the fact that synergy between Notch and other genes is key in understanding how Notch signals contribute to oncogenesis. It remains a remarkable fact that while activating mutations in the Notch receptor have been associated with >50% of T-cell lymphoblastic leukemias (T-ALLs), a search for mutations in other cancers, despite a few suggestive reports, remains essentially unfruitful. Yet, correlative studies have linked Notch activity with a broad spectrum of human cancers and work in mice suggests that while Notch activation promotes proliferation, it is the synergy between Notch and other factors that eventually leads to cancer. Similar synergies have been identified before but the extraordinary complexity of the gene circuitry that modulates the Notch pathway suggests that more such relationships will be uncovered as exemplified by the discovery of Mef2 as a Notch synergistic partner affecting proliferation (Pallavi, 2012).
The transcription factor Mef2 plays an essential role in myogenic differentiation, but several studies have also shown a broad pleiotropic role of Mef2. Mef2 can integrate signals from several signalling cascades through chromatin remodelling factors and other transcriptional regulators to control differentiation events. This study extends the functionality of Mef2 by uncovering the profound effect it can have on proliferation and metastatic cell migration in synergy with Notch signals (Pallavi, 2012).
This is not the first study to link Mef2 with Notch. They have been linked before in the context of myogenesis both in Drosophila and in vertebrates. A ChIP-on-chip analysis of Mef2 target regions identified several Notch pathway components as potential Mef2 targets during Drosophila myogenesis. In human myoblasts, Mef2C was suggested to bind directly to the intracellular domain of Notch via the ankyrin repeat region, suppressing Mef2C-induced myogenic differentiation. Mef2 has also been reported to interact with the Notch coactivator MAML1 and suppress differentiation (Pallavi, 2012).
While upregulation of Mef2 alone does not show overt proliferation effects, these analyses demonstrate that in vivo it can activate MMP1. Even though Mef2 was ectopically expressed in the whole wing pouch, MMP1 expression was confined around the D/V boundary, where endogenous Notch signals are active. This effect of Mef2 overexpression depends on Notch signals, a notion corroborated by the fact that inhibiting Notch activity by RNAi reverses the effects of Mef2 on MMP1 (Pallavi, 2012).
The polarity gene scribble cooperates with Ras signalling to upregulate the JNK pathway, promoting invasiveness and hyperplasticity. However, the synergy seen in this study appears to be scribble independent. The fact that both the scribbled/Ras and the Notch/Mef2 metastatic pathways converge at the level of JNK signal activation suggests that JNK is a crucial regulator of oncogenic behaviour, which is controlled by inputs from multiple signals. Even though there is little evidence that twist activates JNK signalling, it is a crucial regulator of epithelial-to-mesenchymal transition and metastasis and has also been independently linked to both Mef2 and Notch in myogenesis. However, it is noted that the Notch-Mef2 synergy seems to be independent of twist, as Twist cannot replace Mef2 in the synergistic relationship (Pallavi, 2012).
Numerous reports link JNK signalling to normal developmental events requiring cell movement and to metastatic phenomena both in Drosophila and in vertebrates. JNK signals seem to be crucial for controlling gene activities involved in epithelial integrity and the observations from Drosophila suggest that the Nact and Mef2 synergy may be important in JNK-linked carcinogenesis. A role for Notch in controlling JNK signals has been reported previously. While studies carried out using breast cancer samples can only be correlative now, the observations suggest that metastatic breast tumours harbour higher levels of Notch and Mef2 paralogue pairs, consistent with observations in Drosophila (Pallavi, 2012).
Although the majority of studies on Mef2 are focused on muscle development/differentiation, some intriguing links between Mef2C and leukaemias are noteworthy. MEF2C and Sox4 synergize to cause myeloid leukaemia in mice. Analysis of T-ALL patient samples revealed increased levels of Mef2C; however, Mef2C alone could not cause cellular transformation of NIH3T3 cells, but it could do so in the presence of RAS or myc. Given the role of Notch in T-ALL it will be important to examine how activated Notch mutations, often the causative oncogenic mutation, correlate with Mef2 family members. The functional differences between the different Mef2 homologues in humans are not well understood and the specific role each may play in the Notch synergy remains to be elucidated (Pallavi, 2012).
This analysis clearly indicates that, in Drosophila, the underlying molecular mechanism of the Notch/Mef2 synergy relies on the direct upregulation of expression of the prototypical TNF ligand egr through the binding of Mef2 and Su(H), the effector of Notch signals, to regulatory sequences on the egr promoter. In Drosophila, egr is the only JNK ligand while in humans, the superfamily is large and includes the cytokines TNFα (TNF), TRAIL and RANKL which have been associated with tumour progression in numerous human cancers including breast. RANKL plays a key role in bone metastasis of breast cancer, and is the target of a therapeutically effective monoclonal antibody. In breast cancer cells, TNFα, which can signal through several pathways, including JNK and NF-κB, affects proliferation and promotes invasion and metastasis (Pallavi, 2012).
In human breast cancer, clinical relapse after initial treatment is almost always accompanied by metastatic spread and it is almost invariably lethal. ER- tumours tend to respond well to first-line chemotherapy, but a significant subset of these tumours recur. Recurrent ER- tumours are typically resistant to chemotherapy and radiation, and are highly lethal. The current data suggest that ER- tumours that recur but not ER- tumours that do not recur show significant positive correlation between NOTCH1 and all four MEF2 paralogues. Further, the data show that even within the recurrent subset, NOTCH1 expression predicts poor survival but MEF2 expression does not. While these observations do not establish causality, they are consistent with the hypothesis that NOTCH1/MEF2 coexpression identifies a set of breast cancers that are more likely to relapse, and that MEF2 genes act as NOTCH cofactors rather than independently of NOTCH (Pallavi, 2012).
In conclusion, this study in Drosophila uncovers a new functional role for Mef2, which in synergy with Notch affects proliferation and metastasis. Mechanistically, this synergy relies on the direct upregulation of the JNK pathway ligand eiger. The correlation analysis and tumour staining of human cancer samples suggests that the observations in Drosophila may well be valid in humans, defining Notch-Mef2 synergy as a critical oncogenic parameter, one that may be associated with metastatic behaviour, emphasizing the value of model systems in gaining insight into human pathobiology (Pallavi, 2012).
The efficient removal of dead cells is an important process in animal development and homeostasis. Cell corpses are often engulfed by professional phagocytes such as macrophages. However, in some tissues with limited accessibility to circulating cells, engulfment is carried out by neighboring non-professional phagocytes such as epithelial cells. This study investigated the mechanism of corpse clearance in the Drosophila ovary, a tissue that is closed to circulating cells. In degenerating egg chambers, dying germline cells are engulfed by the surrounding somatic follicular epithelium by unknown mechanisms. This study shows that the JNK pathway is activated and required in engulfing follicle cells. The receptor Draper is also required in engulfing follicle cells, and activates the JNK pathway. Overexpression of Draper or the JNK pathway in follicle cells is sufficient to induce death of the underlying germline, suggesting that there is coordination between the germline and follicular epithelium to promote germline cell death. Furthermore, activation of JNK bypasses the need for Draper in engulfment. The induction of JNK and Draper in follicle cells occurs independently of caspase activity in the germline, indicating that at least two pathways are necessary to coordinate germline cell death with engulfment by the somatic epithelium (Etchegaray, 2012).
How non-professional phagocytes respond to dying cells and modulate their phagocytic capabilities is unclear. This study has used the Drosophila ovary as a model to study engulfment by non-professional phagocytes. In this system, the germline can be induced to undergo PCD upon starvation (Etchegaray, 2012).
Following the initiation of PCD, a layer of epithelial FCs synchronously engulfs the dying germline. The engulfment genes drpr, shark and Rac1 are required for engulfment by FCs. The JNK pathway is specifically activated during engulfment and is required for proper engulfment by FCs. This analysis suggests that drpr and JNK are involved in a circuit, where the dying germline activates Drpr, which activates JNK, and JNK signaling leads to an increase in Drpr and likely other engulfment genes. Surprisingly, activation of JNK is sufficient to rescue drpr engulfment defects, indicating that other pathways can carry out engulfment in the absence of drpr. A likely candidate pathway is CED-2, CED-5, CED-12, which can promote engulfment in the absence of ced-1 in C. elegans (Etchegaray, 2012).
In other systems, such as C. elegans and mammalian macrophages, there is redundancy among engulfment pathways. In Drosophila embryos lacking drpr, unprocessed apoptotic particles are detected within glia, suggesting that other pathways can facilitate engulfment of corpses. However, in FCs, drpr is essential for corpse removal. This may be because FCs die if they are engulfment-defective, and there may not be time to activate redundant pathways prior to FC death. It is important to note that drpr (and JNK pathway) mutant FCs survive in healthy egg chambers under starvation conditions; it is only during terminal phases of egg chamber degeneration that they die. Why do the FCs die if they are engulfment defective? Perhaps they have a metabolic requirement, and starve if they cannot obtain nutrients from the germline. Another possibility is that wild-type FCs are programmed to die after completing engulfment, and this PCD may be activated prematurely if engulfment is defective. Mammalian macrophages eliminate themselves after engulfment of specific pathogens or following efferocytosis in ABC transporter mutants. Alternatively, FCs may die because of death 'by confusion', where disruption of the proper signaling network culminates in PCD. Attempts were made to block FC death by expression of caspase inhibitors p35 and Diap1, but FC death was still observed in control, drpr5- and bskDN-expressing egg chambers, indicating that FCs die via a caspase-independent pathway (Etchegaray, 2012).
In mammals, JNK is activated in engulfing professional and non-professional phagocytes, although it remains to be determined whether JNK is required for engulfment. Recently in Drosophila, JNK has been found to be required for the removal of imaginal disc cells succumbing to cell competition, and for the removal of severed axons. These findings suggest that JNK may play a conserved role in engulfment. A role for JNK in engulfment has not been explored in C. elegans and no transcription factor has been shown to activate engulfment genes. This is surprising as levels of CED-1 increase in engulfing cells (Etchegaray, 2012).
How does JNK become activated during engulfment? It may occur via Shark, a kinase that has been shown to interact with both Drpr and JNK in Drosophila. Another candidate is Rac1, which can act upstream of JNK and may act downstream of Drpr. Interestingly, JNK activity is sufficient to restore engulfment in drpr-null egg chambers, suggesting that the primary role of Drpr is to activate JNK. Thus, functions attributed to Drpr such as actin reorganization, Ca2+ signaling, the formation of junctional complexes and autophagy, may depend on JNK activity. Indeed, JNK has been shown to induce autophagy genes in Drosophila (Etchegaray, 2012).
Remarkably, drpr or hepCA overexpression in FCs promoted death of egg chambers even when flies were not starved. This is the first time that overexpression of an engulfment gene has been shown to induce non-autonomous cell death. In other systems, engulfment can promote the death of cells that are weakened, perhaps on the brink of death. For example, mutations in engulfment genes can lead to the survival of cells fated to die in C. elegans ced-3 hypomorphs, and to the survival of 'loser' cells in Drosophila imaginal discs. In mammals, neuronal exposure to amyloid Aβ peptide or LPS leads to cell death, which can be inhibited by blocking phagocytosis. Interestingly, treated neurons transiently expose phosphatidylserine, perhaps to announce their vulnerability. These findings differ from scenarios in that the egg chambers are healthy. However, mid-stage egg chambers are more susceptible to death stimuli than egg chambers at other stages of oogenesis. Overexpression of Drpr in early oogenesis did not lead to egg chamber death, but death was observed later in mid-oogenesis. Thus, it may be that Drpr is not sufficient to kill the germline until mid-oogenesis, when it is more vulnerable. The factors that contribute to this vulnerability are unknown (Etchegaray, 2012).
Overexpression of drpr in FCs led to death of the underlying NCs before there was any engulfment by the FCs, suggesting that drpr produces a death signal that is sent to the germline. Overexpression of the JNKK hepCA led to destruction of egg chambers earlier in oogenesis than overexpression of drpr, suggesting that JNK did not require the vulnerability at midoogenesis. Furthermore, hepCA-expressing FCs engulfed intact NCs ('hyper-engulfment'), rather than inducing death first. This phenotype resembles the process of entosis, where living cells are engulfed by their neighbors (Etchegaray, 2012).
Germline PCD in mid-oogenesis requires caspases, and the current results indicate that caspase activity is required to stimulate FCs to engulf the germline. Surprisingly, germline caspase activity was not necessary or sufficient to activate JNK or induce Drpr in the FCs. This suggests that a caspase-dependent pathway, distinct from the pathway(s) that activate Drpr-JNK, is required for engulfment in mid-oogenesis. The caspase-dependent signal and the responding pathway in the FCs remain to be elucidated. Another open issue is how Drpr, and thereby JNK, becomes activated in response to the dying germline. The complexity of cell surface modifications that occur during apoptosis will make this a challenge to determine. Draper and JNK may become activated directly in the FCs in response to starvation; however, this scenario seems less likely than activation by the dying germline for two reasons. First, many egg chambers do not die immediately upon starvation, and activation of Draper and JNK was observed only in egg chambers that had begun to die. Second, germline death triggered by overexpression of dcp-1 could lead to Draper and JNK activation in FCs in the absence of starvation. The activation of JNK and Drpr illustrate ways in which non-professional phagocytes change in response to apoptotic cells. Future work will reveal the network of pathways activated in non-professional phagocytes to enhance apoptotic cell clearance (Etchegaray, 2012).
Two extraembryonic tissues form early in Drosophila development. One, the amnioserosa, has been implicated in the morphogenetic processes of germ band retraction and dorsal closure. The developmental role of the other, the yolk sac, is obscure. By using live-imaging techniques, intimate interactions are reported between the amnioserosa and the yolk sac during germ band retraction and dorsal closure. These tissue interactions fail in a subset of myospheroid (mys: ßPS integrin) mutant embryos, leading to failure of germ band retraction and dorsal closure. The Drosophila homolog of mammalian basigin (EMMPRIN , CD147) -- an integrin-associated transmembrane glycoprotein -- is highly enriched in the extraembryonic tissues. Strong dominant genetic interactions between basigin and mys mutations cause severe defects in dorsal closure, consistent with basigin functioning together with ßPS integrin in extraembryonic membrane apposition. During normal development, JNK signaling is upregulated in the amnioserosa, as midgut closure disrupts contact with the yolk sac. Subsequently, the amnioserosal epithelium degenerates in a process that is independent of the reaper, hid, and grim cell death genes. In mys mutants that fail to establish contact between the extraembryonic membranes, the amnioserosa undergoes premature disintegration and death. It is concluded that intimate apposition of the amnioserosa and yolk sac prevents anoikis of the amnioserosa. Survival of the amnioserosa is essential for germ band retraction and dorsal closure. It is hypothesized that during normal development, loss of integrin-dependent contact between the extraembryonic tissues results in JNK-dependent amnioserosal disintegration and death, thus representing an example of developmentally programmed anoikis (Reed, 2004).
Physical interaction of the amnioserosa and yolk sac has been shown to play a crucial role in both germ band retraction and dorsal closure of the embryo. βPS integrin mediates extraembryonic membrane interactions that are required for survival of the amnioserosa. Anoikis of the amnioserosa occurs during normal development after closure of the midgut disrupts integrin-dependent apposition of the amnioserosa and yolk sac. In mys mutants, failure to establish apposition of extraembryonic membranes leads to premature anoikis of the amnioserosa. A possible role for JNK signaling and the reaper/hid/grim cell death genes in amnioserosal anoikis during normal development was investigated (Reed, 2004).
It is possible to visualize a subset of the amnioserosal cells as acridine orange positive either before they leave the tube or shortly thereafter. Both acridine orange staining and engulfment by hemocytes are hallmarks of dying cells. To determine whether death of amnioserosal cells might be reaper dependent, it was asked whether reaper expression could be visualized in the amnioserosal cells prior to or after extrusion. No reaper-expressing cells were detected. To further test whether amnioserosal cell death might be reaper dependent, the H99 deficiency [Df(3L)H99] was used; this deficiency removes the reaper, head involution defective (hid), and grim genes, and the amnioserosa with anti-HNT antibody was visualized. If amnioserosal death were reaper dependent, one would expect HNT-positive cells to persist in H99 mutants when compared with wild-type. Such persistence does not occur. While it is conceivable that HNT expression is downregulated in a persistent amnioserosa, the simplest interpretation of these data is that death of the amnioserosa is reaper independent. This conclusion is consistent with the recent suggestion that Drosophila embryos have a caspase-independent cell engulfment system, which is still operative in H99 mutants (Reed, 2004).
It has been shown that loss of integrin-dependent contact between cells and the extracellular matrix leads to cell death, a process referred to as anoikis. Anoikis is promoted by the Jun amino-terminal kinase (JNK) pathway. Previous analyses have shown that JNK signaling in the amnioserosa is downregulated prior to dorsal closure. In those analyses, puckered-lacZ expression was used as a read-out of JNK signaling, and it was shown that relocation of JUN and FOS proteins from the nucleus to the cytoplasm of amnioserosal cells correlates with downregulation of JNK signaling. While JNK signaling is downregulated in the amnioserosa prior to dorsal closure, JNK signaling is upregulated in this tissue as dorsal closure approaches completion. Thus, reactivation of JNK signaling in the amnioserosa follows loss of integrin-dependent apposition of the amnioserosa and yolk sac membrane and precedes amnioserosal disintegration and death. These data are consistent with the hypothesis that midgut closure disrupts integrin-dependent apposition of the amnioserosa and yolk sac, thus inducing JNK signaling in the amnioserosa and its subsequent anoikis (Reed, 2004).
It remains to be determined whether disintegration and death of the amnioserosa during normal development is caused solely by loss of contact with the yolk sac (i.e., is nonautonomously induced) versus whether signals from cell types other than the yolk -- or even an amnioserosa-autonomous program -- also play a role. For example, it is possible that upregulation of JNK signaling in the amnioserosa is independent of loss of contact with the yolk sac. Analysis of mutants lacking a midgut provide a test of this possibility: if disintegration and death of the amnioserosa occur even when apposition with the yolk sac is maintained, signals from other cell types or amnioserosa-autonomous processes would be implicated (Reed, 2004).
The specific role of JNK signaling in amnioserosal anoikis is difficult to assess because downregulation of JNK signaling in the amnioserosa and up-regulation of JNK signaling in the leading edge of the epidermis are required for dorsal closure. Thus JNK pathway mutants stall morphogenesis prior to dorsal closure, making it impossible to assess a possible later role. Expression of dominant-negative JNK specifically in the amnioserosa only later in development, when closure is almost complete, will be necessary to rigorously test the role of JNK activation in amnioserosal anoikis (Reed, 2004).
It is concluded that the extraembryonic tissues of Drosophila play a crucial role in directing embryonic morphogenesis. Close apposition of the yolk sac membrane and the basal cell membranes of the amnioserosa is dependent on βPS integrin. This intimate membrane association is required to promote survival and to prevent anoikis of the amnioserosa. The amnioserosa then directs germ band retraction and dorsal closure through physical contacts and/or signaling. Disintegration and death of the amnioserosa after closure of the epidermis and midgut correlates with upregulation of JNK signaling in the amnioserosa, is independent of reaper/hid/grim function, and is likely to represent the first example of developmentally programmed anoikis in Drosophila (Reed, 2004).
In many metazoans, damaged and potentially dangerous cells are rapidly eliminated by apoptosis. In Drosophila, this is often compensated for by extraproliferation of neighboring cells, which allows the organism to tolerate considerable cell death without compromising development and body size. Despite its importance, the mechanistic basis of such compensatory proliferation remains poorly understood. Apoptotic cells are shown to express the secretory factors Wingless and Decapentaplegic. When cells undergoing apoptosis were kept alive with the caspase inhibitor p35, excessive nonautonomous cell proliferation is observed. Significantly, Wg signaling is necessary and, at least in some cells, also sufficient for mitogenesis under these conditions. Finally, evidence is provided that the DIAP1 antagonists reaper and hid can activate the JNK pathway and that this pathway is required for inducing wg and cell proliferation. These findings support a model where apoptotic cells activate signaling cascades for compensatory proliferation (Ryoo, 2004).
To investigate how the inhibition of diap1 may lead to mitogen expression, attention was focused on Dronc and the Jun N-terminal Kinase (JNK) pathway. Dronc has been implicated in compensatory proliferation, and its activity can be inhibited by the expression of droncDN. In addition, the JNK signaling pathway was considered as a candidate, since its activity is known to correlate with many forms of stress-provoked apoptosis, including disruption of morphogens, cell competition, and rpr expression. In Drosophila, the JNK pathway can be effectively blocked by the expression of puckered (puc), which encodes a phosphatase that negatively regulates JNK (Ryoo, 2004).
To induce patches of undead cells, wing imaginal discs were generated with mosaic clones expressing hid and p35. 48 hr after induction, these imaginal discs contained hid-expressing clones that autonomously induced wg. Using this experimental setup, it was asked whether additional expression of either droncDN or puc would block wg induction in undead cells. When droncDN was coexpressed, a subset of the hid-expressing population was still able to induce wg. In contrast, when puc was coexpressed, wg induction by hid was almost completely blocked. These results provide evidence that the JNK pathway is required for wg induction under these conditions but fail to uncover a similar requirement for Dronc (Ryoo, 2004).
To independently investigate the role of puc and droncDN in compensatory proliferation, the size of wing discs harboring undead cells was measured and they were compared with those of the sibling controls. Under the experimental conditions, wing discs harboring hid- and p35-expressing clones were on average 53% larger than their sibling controls. Coexpression of puc within these undead clones significantly limited growth, resulting in only a small increase in wing disc size that was not statistically significant. In contrast, coexpression of droncDN did not limit growth. Wing size measurements also correlated with the degree of wg induction. The larger size of discs harboring hid- and p35-expressing cells is not due simply to extra cell survival: (1) these undead cells are derived from the normal lineage; (2) the size of wing discs expressing hid, p35, and puc serves as a control. In this case, although a large number of undead cells were generated, no significant increase in disc size was observed, in stark contrast to the discs expressing hid and p35 only. It is concluded that the JNK pathway is required for the nonautonomous growth promoting activity of the undead cells (Ryoo, 2004).
To confirm a role of puc in imaginal disc growth, rpr and p35 werecoexpressed in wild-type and puc−/+ imaginal discs. Like hid, rpr is a DIAP1 antagonist, but with a weaker cell killing activity when overexpressed in imaginal disc cells. In a puc+/+ background, a small amount of ectopic wg expression was observed, indicative of rpr's weaker DIAP1 inhibiting activity. In contrast, ectopic wg expression was strongly enhanced in puc−/+ discs. Because the puc allele used, pucE69, also acts as a lacZ reporter, JNK pathway induction could be monitored simultaneously. wg induction in undead cells correlates very well with puc-lacZ expression, with a stronger induction at the center of the wing pouch. These results further support the role of JNK in the induction of wg (Ryoo, 2004).
Next to be tested was whether the reduction of puc had an effect on apoptosis-induced cell proliferation. Whereas puc−/+ discs expressing only p35 had BrdU incorporation similar to wild-type discs, coexpression of rpr and p35 in puc−/+ led to a significant increase in BrdU incorporation. Also, the size of these discs were on average 41% larger than those coexpressing rpr and p35 in a puc+/+ background. Taken together, these results show that diap1 inhibition leads to JNK activation and that JNK activity promotes wg induction and cell proliferation (Ryoo, 2004).
To directly test if JNK signaling can activate wg and dpp expression, hepCA, a constitutively active form of hemipterous (hep), the Drosophila JNK kinase was conditionally expressed. Expression of hepCA causes induction of wg-lacZ within 22 hr and to a lesser extent also dpp-lacZ. These ß-gal-expressing cells shifted basally and were apoptotic as assayed by anti-active caspase-3 antibody labeling. Hid protein levels were also elevated in these cells. Significantly, since p35 was not use to block apoptosis in this experiment, this demonstrates that wg and dpp can be induced not only in undead cells, but also in 'real' apoptotic cells (Ryoo, 2004).
This study provides evidence that the central apoptotic regulators can control the activity of mitogenic pathways. In particular, inhibition of DIAP1, either via expression of Reaper and Hid or by mutational inactivation, leads to the induction of the putative mitogens wg and dpp. When apoptosis was initiated through DIAP1 inhibition but cells were kept alive by blocking caspases, the resulting 'undead cells' exhibited strong mitogenic activity and stimulated tissue overgrowth. Inhibiting wg signaling with a conditional TCFDN blocked cell proliferation in imaginal discs, indicating that wg has an essential mitogenic function. Finally, evidence was provided that the JNK pathway mediates mitogen expression and imaginal disc overgrowth in response to rpr and hid. Based on these results, it is proposed that apoptotic cells actively signal to induce compensatory proliferation. DIAP1 inhibits both caspases as well as dTRAF1. According to this model, when DIAP1 is inhibited in response to cellular injury, the JNK pathway is activated and wg/dpp are induced in apoptotic cells. Secretion of these factors stimulates growth of proliferation-competent neighboring cells and leads to compensatory proliferation (Ryoo, 2004).
When cells undergo apoptosis, they can stimulate the proliferation of nearby cells, a process referred to as compensatory cell proliferation. The stimulation of proliferation in response to tissue damage or removal is also central to epimorphic regeneration. The Hippo signaling pathway has emerged as an important regulator of growth during normal development and oncogenesis from Drosophila to humans. This study shows that induction of apoptosis in the Drosophila wing imaginal disc stimulates activation of the Hippo pathway transcription factor Yorkie in surviving and nearby cells, and that Yorkie is required for the ability of the wing to regenerate after genetic ablation of the wing primordia. Induction of apoptosis activates Yorkie through the Jun kinase pathway, and direct activation of Jun kinase signaling also promotes Yorkie activation in the wing disc. It was also shown that depletion of neoplastic tumor suppressor genes, including lethal giant larvae and discs large, or activation of aPKC, activates Yorkie through Jun kinase signaling, and that Jun kinase activation is necessary, but not sufficient, for the disruption of apical-basal polarity associated with loss of lethal giant larvae. These observations identify Jnk signaling as a modulator of Hippo pathway activity in wing imaginal discs, and implicate Yorkie activation in compensatory cell proliferation and disc regeneration (Sun, 2011).
Many tissues have the capacity respond to the removal or death of cells by increasing proliferation of the remaining cells. In Drosophila, this phenomenon has been characterized both in the context of imaginal disc regeneration and compensatory cell proliferation. These studies implicate the Hippo signaling pathway as a key player in these proliferative responses to tissue damage. After genetically ablating the wing primordia by inducing apoptosis, it was observed that Yki becomes activated to high levels in surrounding cells, based on its nuclear abundance and induction of a downstream target of Yki transcriptional activity. Moreover, high level Yki activation is crucial for wing disc regeneration, as even modest reduction of Yki levels, to a degree that has only minor effects on normal wing development, severely impaired wing disc regeneration. While it was known that Yki is required for wing growth during development, the current observations establish that Yki is also required for wing growth during regeneration, and moreover that regeneration requires higher levels of Yki activation than during normal development (Sun, 2011).
These studies identify Jnk activation as a promoter of Yki activity in the wing disc. Most aspects of imaginal disc development, including imaginal disc growth, normally do not require Jnk signaling. By contrast, Jnk signaling is both necessary and sufficient for Yki activation in response to wing damage. Jnk signaling has previously been linked to compensatory cell proliferation and regeneration in imaginal discs, and it is now possible to ascribe at least part of that requirement to activation of Yki. However, Jnk signaling also promotes the expression of other mitogens, including Wg, which were linked to regeneration and proliferative responses to apoptosis. Wg and Yki are not required for each other's expression, suggesting that they are regulated and act in parallel to influence cell proliferation after tissue damage. The mechanism by which Jnk activation induces Yki activation is not yet known. The observation that it could be suppressed by over-expression of Wts or Hpo suggests that it might impinge on Hippo signaling at or upstream of Hpo and Wts, but the possibility that Jnk-dependent Yki regulation occurs in parallel to these Hippo pathway components cannot be excluded. The high level of nuclear Yki localization is striking by contrast with the more modest effects of upstream tumor suppressors in the Hippo pathway, which suggests that Jnk might regulate Yki through a distinct mechanism, or simultaneously affect multiple upstream regulators (Sun, 2011).
Strong Yki activation was detected within the wing and haltere discs in response to Jnk activation, but weaker or non-existent effects in leg or eye discs. Jnk activation has previously been linked to oncogenic effects of neoplastic tumor suppressors in eye discs, and it is possible that Yki activation might be induced in eye discs if a distinct Jnk activation regime were employed. Nonetheless, since identical conditions were employed in both wing and eye discs, isolating them from the same animals, these studies emphasize the importance of context-dependence for Yki activation by Jnk. A link between Jnk activation and Yki activation is not limited to the wing however, as a connection between these pathways was recently discovered in the adult intestine, where damage to intestinal epithelial cells, and activation of Jnk, can activate also Yki (Sun, 2011).
There was a general correspondence between activation of Jnk and activation of Yki under multiple experimental conditions, including expression of Rpr, direct activation of Jnk signaling by Egr or Hep.CA (an activated form of the Jnk kinase Hemipterous), and depletion of lgl. Some experiments, most notably direct activation of Jnk by Hep.CA, revealed a non-autonomous effect on Yki, which could imply that the influence of Jnk on Yki activity is indirect. Although the basis for this non-autonomous effect is not yet known, the hypothesis that it is actually also mediated through Jnk signaling is favored, since it has been reported that Jnk activation can propagate from cell to cell in the wing disc. Consistent with this possibility, a non-autonomous activation of Jnk adjacent to lgl depleted cells was seen to be blocked by depletion of bsk solely within the lgl RNAi cells. Conversely, alternative signals previously implicated in compensatory cell proliferation do not appear to be good candidates for mediating Yki activation, since it was found that Wg is not required for Yki activation in regenerating discs, and prior studies did not detect a direct influence of Dpp pathway activity on Yki activation (Sun, 2011).
Activation of Yki adjacent to Egr- or Rpr-expressing cells was also reduced by over-expression of Wts. This might reflect an influence of Yki on signaling from these cells, but because expression of Wts inhibits Yki activity, and activated Yki promotes expression of an inhibitor of apoptosis (Diap1), it is also possible that this effect could be explained simply by Wts over-expression resulting in reduction or more rapid elimination of Egr- or Rpr-expressing cells; the reduced survival of these cells would then limit their ability to signal to neighbors (Sun, 2011).
Although Jnk has been implicated in compensatory cell proliferation and regeneration, it is better known for its ability to promote apoptosis. The dual, opposing roles of Jnk signaling as a promoter of apoptosis and a promoter of cell proliferation raise the question of how one of these distinct downstream outcomes becomes favored in cells with Jnk activation (see Diverse inputs and outputs of Jnk signaling). Given the links between Jnk activation and human diseases, including cancer, defining mechanisms that influence this is an important question, and the identification of the role of Yki activation in Jnk-mediated proliferation and wing regeneration should facilitate future investigations into how the balance between proliferation or apoptosis downstream of Jnk is regulated (Sun, 2011).
Hippo signaling is regulated by proteins that exhibit discrete localization at the subapical membrane, e.g., Fat, Ex, and Merlin. The observation that disruption of apical-basal polarity is associated with disruption of Hippo signaling underscores the importance of this localization to normal pathway regulation. These observations establish that Hippo signaling is inhibited by neoplastic tumor suppressor mutations, resulting in Yki activation, and that this activation of Yki is required for the tumorous overgrowths associated with these mutations (Sun, 2011).
Although these results agree with these recent studies in linking lgl to Hippo signaling (Grzeschik, 2010), there are some notable differences. A previous study examined lgl mutant clones in the eye imaginal disc, under conditions where cells retained apical-basal polarity, whereas this study examined wing imaginal discs, where apical-basal polarity was lost. Intriguingly this study found that conditions associated with activation of Yki by Jnk in the wing disc were not sufficient to activate Yki in the eye disc. This observation, together with the discovery that loss of polarity in lgl depleted wing cells requires Jnk activation, suggests as a possible explanation for why lgl null mutant clones retain apical-basal polarity in eye discs, that eye disc cells have a distinct, and apparently reduced, sensitivity to Jnk activation as compared to wing disc cells (Sun, 2011).
This study also identified distinct processes linked to Yki activation in the absence of lgl. A previous study reported an effect of lgl on Hpo protein localization (Grzeschik, 2010). In wing discs, the discrete apical localization of Hpo was observed in studies of eye discs. Thus, the proposed mechanism, involving activation of Yki via mis-localization of Hpo and dRassf, might not be relevant to the wing. By contrast, this study identified an essential role for Jnk signaling in regulating Yki activation in lgl-depleted cells in the wing. Because this study did not detect an effect of direct Jnk activation on Yki in eye discs, it is possible that Lgl can act through multiple pathways to influence Yki, including a Jnk-dependent pathway that is crucial in the wing disc, and a Jnk-independent pathway that is crucial in the eye disc. Grzeschik (2010) also linked the influence of lgl in the eye disc to its antagonistic relationship with aPKC. The observation that the influence of aPKC in the wing depends on Jnk activation is consistent with an Lgl-aPKC link, and identifies a role for Jnk activation in the oncogenic effects of aPKC (Sun, 2011).
The observation that the loss of polarity in lgl RNAi discs is dependent upon Jnk signaling was unexpected, but a related observation was recently reported by Zhu; 2010). These results suggest that the established role of the Lgl-Dlg-Scrib complex in maintaining epithelial polarity depends in part on repressing Jnk activity. However, since Jnk activation on its own was not sufficient to disrupt polarity, multiple polarity complexes might need to be disturbed in order for wing cells to lose apical-basal polarity, including both Lgl and additional, Jnk-regulated polarity complexes (Sun, 2011).
The discovery of the role of Jnk signaling in Yki activation provides a common molecular mechanism for the overgrowths observed in conjunction with mutations of neoplastic tumor suppressors, and those associated with compensatory cell proliferation, because in both cases a proliferative response is mediated through Jnk-dependent activation of Yki. Although the molecular basis for the linkage of these two pathways is not understood yet, it operates in multiple Drosophila organs, and thus appears to establish a novel regulatory input into Hippo signaling that is of particular importance in abnormal or damaged tissues. Moreover, Jnk activation has also been observed in conjunction with regeneration of disc fragments after surgical wounding, and thus its participation in regeneration is not limited to paradigms involving induction of apoptosis. It is also noteworthy that under conditions of widespread lgl depletion (i.e., lgl mutant or lgl RNAi), and consequent Jnk activation, the balance between induction of apoptosis and induction of cell proliferation is shifted towards a proliferative response. By contrast, in the wing disc clones of cells mutant for lgl fail to survive, unless oncogenic co-factors are co-expressed. The loss of lgl mutant clones in wing discs was recently attributed to cell competition. Together, these observations suggest that the choice between proliferative versus apoptotic responses to Jnk activation can be influenced by the Jnk activation status of neighboring cells (Sun, 2011).
JNK signaling functions to induce defense mechanisms that protect organisms against acute oxidative and xenobiotic insults. Using Drosophila as a model system, the role of autophagy was investigated as such a JNK-regulated protective mechanism. Oxidative stress was shown to induce autophagy in the intestinal epithelium by a mechanism that requires JNK signaling. Consistently, artificial activation of JNK in the gut gives rise to an autophagy phenotype. JNK signaling can induce the expression of several autophagy-related (ATG) genes, and the integrity of these genes is required for the stress protective function of the JNK pathway. In contrast to autophagy induced by oxidative stress, non-stress related autophagy, as it occurs for example in starving adipose or intestinal tissue, or during metamorphosis, proceeds independently of JNK signaling. Autophagy thus emerges as a multifunctional process that organisms employ in a variety of different situations using separate regulatory mechanisms (Wu, 2009).
Much interest has focused on autophagy as a mechanism by which cells defend themselves against environmental stresses. The notion that autophagy can have cell protective functions first emerged based on the finding that adaptations of several organisms to unfavorable environmental conditions require ATG genes and autophagy. Examples range from sporulation in yeast to the formation of fruiting bodies in Dictyostelium and dauer larvae in Caenorabditis elegans. Autophagy can also confer resistance to oxidative stress. Mutations that compromise the autophagy system result in increased stress sensitivity. Drosophila loss-of-function mutants for ATG7 or ATG8a, for example, are hypersensitive to H2O2 (Juhasz, 2007a; Simonsen, 2008). Cells can respond to a variety of insults, including oxidative stress, with increased autophagic activity. However, how the autophagy machinery senses and responds to stress is not thoroughly understood. Such regulation could occur at several levels, as autophagy can be regulated by transcriptional, as well as post-transcriptional mechanisms. Consistent with a function of gene regulation in this context, multiple reports show that ATG gene expression can be stimulated in response to stresses (Wu, 2009).
This work explores the control of autophagy by JNK signaling in Drosophila. The JNK pathway is an evolutionarily conserved signal transduction system that can be triggered by several types of external insults, including oxidative stress. Stress signals are conveyed by a MAP kinase cascade, which in Drosophila, consists of one of several JNKKKs (Jun kinase kinase kinases), a JNKK, the MKK7 ortholog Hemipterous (Hep) and the JNK, Basket (Bsk). The duration and extent of JNK responses is tightly controlled and restricted in time and space by a number of negative feedback mechanisms. One of these mechanisms relies on the transcriptional activation of the JNK specific MAP kinase phosphatase puckered (puc) (Wu, 2009).
JNK signaling has been implicated in the regulation of a range of cellular stress responses. These include the induction of antioxidant and repair programs, and, depending on the nature of the inducing signal and the cell type, apoptosis. Such responses can be orchestrated by changes in gene expression mediated by several transcription factors that are regulated by JNK signaling. In addition, JNK can directly regulate cell responses such as apoptosis by phosphorylating effector molecules (Wu, 2009).
It as been show that activation of the JNK pathway can protect fruit flies against oxidative toxicity. For example, flies in which JNK signaling is elevated due to the loss of one copy of the gene encoding the JNK phosphatase Puckered, or by over expressing the JNK kinase Hep, gain resistance to the free radical inducing drug paraquat (Wang, 2003; Wang, 2005; Wu, 2009).
JNK signaling emerges as a regulator of multiple mechanisms that cells can engage to increase resistance against external stresses. Recent studies in cell culture models indicate that autophagy is one of these responses. This study shows that autophagy is part of a JNK-induced stress defense program in Drosophila and explores the relationship between stress-induced autophagy and autophagy that is regulated in response to metabolic or developmental signals (Wu, 2009).
Autophagy is now recognized as a process that has multiple functions in addition to balancing energy homeostasis. It has become increasingly apparent that cells also rely on autophagy for protecting themselves against a battery of potentially harmful insults. For example, autophagy can contribute to cellular defenses against pathogens, bacterial toxins, oxidative stress and ER stress (Wu, 2009).
To explore the function and regulation in organismic stress responses, the Drosophila intestinal epithelium was studied. This tissue is directly exposed and potentially vulnerable to oxidative stress in the form of dietary toxicants or of H2O2 that can be internally generated as part of pathogen defenses. It can therefore be expected that the gut employs potent defense and regeneration systems. Exposure of the Drosophila intestine to oxidative stress, or deliberate activation of JNK signaling in the gut epithelium, results in a prominent rise of autophagosome density as monitored by Lysotracker red or GFP-LC3 staining. This effect resembles the well-established induction of autophagy in this and other tissues in response to starvation. Ultrastructure analysis by transmission electron microscopy confirms that JNK can effectively induce the formation of bona fide autophagosomes. The combined evidence from histology and microscopic analyses, the genetic interactions between JNK and ATG genes, and the induction of ATG gene expression by JNK, support the conclusion that JNK and oxidative stress can induce autophagy in the Drosophila gut (Wu, 2009).
The data suggest that JNK signaling induces autophagy, at least in part, by transcriptional activation of ATG genes. Such a mechanism would be consistent with several previous reports indicating that conditions that stimulate autophagy, such as starvation and stress, also lead to increased expression levels of ATG genes. Furthermore, the deliberate expression of ATG1, ATG6 or ATG8a by itself is sufficient to drive cells into autophagy. It is therefore plausible that the JNK-induced increases in ATG gene expression levels observed in this study might drive and/or sustain autophagy in stressed organs. However, it is also clear that autophagy can be controlled by mechanisms other than gene expression. For instance, protein phosphorylation, lipidation, and processing events have been shown to regulate the process. Two recent studies conducted in mammalian cell lines indicate that JNK can induce autophagy by phosphorylating Bcl2, thereby relieving its inhibitory effect on Beclin 1, the ATG6 homolog (Pattingre, 2008; Wei, 2008). It thus emerges that JNK may impinge on autophagy at multiple regulatory levels. Such a scenario bears an interesting resemblance of the better-understood role of JNK in the regulation of apoptosis, a process that it can control by transcriptional, as well as non-transcriptional mechanisms. It is at present a matter of speculation how these different layers of regulation are integrated and how they may have evolved. In this regard it is interesting that the only anti-death Bcl2 family member in Drosophila, the buffy gene product, does not contain JNK phosphorylation sites, suggesting that Bcl2-dependent mechanisms do not contribute to JNK induced autophagy in Drosophila. It is possible that in flies JNK acts predominantly at the transcription level, and that the role of Bcl2 in this context has evolved later (Wu, 2009).
The oxidative stress hypothesis of aging predicts that bolstering resistance against oxidative damage can extend longevity of organisms, and it has been shown previously that JNK signaling can control aging by deploying cellular oxidative stress defenses (Wang, 2003). The data presented in this study, that indicate that JNK-mediated induction of autophagy can increase oxidative stress resistance, therefore raise the question of whether such a mechanism might be also relevant for the regulation of longevity. Interestingly, several published studies support the conclusion that ATG genes, and by extension the process of autophagy, are required for the lifespan extending effects of caloric restriction or reduced Tor signaling. A recent study even finds that over expression of ATG8a under the control of one particular neuron specific promoter is sufficient to extend lifespan and confer stress resistance in Drosophila (Simonsen, 2008; Wu, 2009 and references therein).
The downstream transcription factor(s) that mediate the activation of ATG genes in response to JNK signaling are not known at this point. However, recent experiments by Juhasz indicate that the transcription factor FoxO is required for the induction of autophagy in flies that have been deprived of food (Juhasz, 2007b). In mammals it has been shown that the FoxO can induce ATG gene expression. Drosophila FoxO to be critical for JNK-mediated stress resistance. FoxO is therefore a good candidate to execute the transcriptional activation of ATG gene expression in response to JNK signaling. Further experiments are required to determine the mechanisms by which the transcriptional regulation of autophagy proceeds (Wu, 2009).
The findings presented in this study indicate that the diverse cues that can cause a cell to undergo autophagy, including metabolic, hormonal and stress signals, are transmitted by distinct signaling systems. While the JNK pathway induces autophagy in response to oxidative stress, changes in PI3K and Tor pathways stimulate autophagy in response to food deprivation and ecdysone in a JNK-independent manner. Consistent with the conclusion that the induction of autophagy in conditions of limited food supply does not involve the JNK signaling pathway, no activation of a JNK reporter gene was detected in gut cells under the starvation conditions employed in this study, which nevertheless effectively induce autophagy. However, protracted or extreme starvation may derail vital cell functions causing stress and a JNK response. For example, mammalian cells that are cultured in nutrient-deprived media activate JNK and consequently autophagy (Wu, 2009).
The induction of autophagy by oxidative stress so far discussed contrasts with developmentally programmed autophagy as it prominently occurs during metamorphosis in the Drosophila fat body. This mechanism is hormonally regulated by the ecdysone system and does not appear to require JNK (Wu, 2009).
Further genetic and molecular studies on the complex regulation of autophagy in different biological settings and organ systems are a high priority. Insight into the pleiotropic functions of this process will be valuable not only for developmental and cell biology, but also for the understanding of pathologies that are correlated with oxidative damage to cells and tissues, such as aging related and degenerative diseases (Wu, 2009).
The Unfolded Protein Response of the endoplasmic reticulum (UPRER: see Drosophila Inositol-requiring enzyme-1) controls proteostasis by adjusting the protein folding capacity of the ER to environmental and cell-intrinsic conditions. In metazoans, loss of proteostasis results in degenerative and proliferative diseases and cancers. The cellular and molecular mechanisms causing these phenotypes remain poorly understood. This study shows that the UPRER is a critical regulator of intestinal stem cell (ISC) quiescence in Drosophila melanogaster. ISCs were found to require activation of the UPRER for regenerative responses, but a tissue-wide increase in ER stress was found to trigger ISC hyperproliferation and epithelial dysplasia in aging animals. These effects are mediated by ISC-specific redox signaling through Jun-N-terminal Kinase (JNK) and the transcription factor CncC. The results identify a signaling network of proteostatic and oxidative stress responses that regulates ISC function and regenerative homeostasis in the intestinal epithelium (Wang, 2014 PubMed).
Caspase-independent cell death is known to be important in physiological and pathological conditions, but its molecular regulation is not well-understood. Eiger is the sole fly ortholog of TNF. The ectopic expression of Eiger in the developing eye primordium caused JNK-dependent but caspase-independent cell death. To understand the molecular basis of this Eiger-induced nonapoptotic cell death, a large-scale genetic screen was performed in Drosophila for suppressors of the Eiger-induced cell death phenotype. It was found that molecules that regulate metabolic energy production are central to this form of cell death: it was dramatically suppressed by decreased levels of molecules that regulate cytosolic glycolysis, mitochondrial β-oxidation of fatty acids, the tricarboxylic acid cycle, and the electron transport chain. Importantly, reducing the expression of energy production-related genes did not affect the cell death triggered by proapoptotic genes, such as reaper, hid, or debcl, indicating that the energy production-related genes have a specific role in Eiger-induced nonapoptotic cell death. It was also found that energy production-related genes regulate the Eiger-induced cell death downstream of JNK. In addition, Eiger induced the production of reactive oxygen species in a manner dependent on energy production-related genes. Furthermore, this cell death machinery was shown to be involved in Eiger's physiological function, because decreasing the energy production-related genes suppressed Eiger-dependent tumor suppression, an intrinsic mechanism for removing tumorigenic mutant clones from epithelia by inducing cell death. This result suggests a link between sensitivity to cell death and metabolic activity in cancer (Kanda, 2011).
At least some nonapoptotic cell deaths can be categorized as necroptosis, in which cells undergo nonapoptotic cell death under apoptosis-deficient conditions when treated with agonistic ligands of death receptors, such as TNFα, FasL, or TRAIL (Christofferson, 2010; see The signaling complexes induced by TNFα to mediate NF-kappaB activation, apoptosis and necroptosis.). The Eiger-induced cell death shares features with necroptosis in that it is triggered by TNF family proteins, produces ROS, and is caspase-independent. Furthermore, the Drosophila homolog of a tumor suppressor protein, Cylindromatosis, one of the essential regulators of necroptosis (Wang, 2008; Hitomi, 2008), has been shown to regulate JNK activation in Eiger-induced cell death signaling (Xue, 2007). However, despite the high conservation of most of the apoptotic machinery, blast search analysis has not identified Drosophila homologs of receptor-interacting protein (RIP) 1 or RIP3, the essential kinases for inducing necroptosis (Kanda, 2011 and references therein).
Oxidative stress could be induced downstream of the activated JNK pathway. However, it was also found that the knockdown of energy production-related genes such as cyt.c-d or GAPDH2 did not suppress the dTAK1- or HepCA-induced cell death phenotype. This finding could be caused by the overexpression of dTAK1- or HepCA-induced additional pathways, because dTAK1 or HepCA overexpression were found to induced JNK activation much more strongly than Eiger overexpression. Because dTAK1 also functions downstream of Imd in the innate immune response, Imd-related signaling was another possible mechanism for mediating Eiger signaling. Therefore, the genetic interaction between Eiger-induced cell death and Imd or Imd-related genes was examined. However, no significant interactions between them were found. Therefore, it would be interesting to examine if other proteins can substitute for the functions of RIP1 or RIP3 in Eiger signaling or if fly TNF signaling uses other mechanisms to induce nonapoptotic cell death (Kanda, 2011).
It has been reported that TNF-induced nonapoptotic cell death leads to the RIP3-dependent activation of glycogen phosphorylase, which is the rate-limiting enzyme in the degradation of glycogen and therefore, the key molecule for regulating energy production (Zhang, 2009). In this context, ROS can be generated by the production of excess energy. This finding could explain why the down-regulation of energy production-related genes suppressed Eiger-induced cell death. However, it was also observed that the amount of ATP in the eye antenna imaginal disc decreased when Eiger was overexpressed, and this decrease in ATP was cancelled by the knockdown of bsk, CPTI, pgk, or cyt.c-d. This finding suggests that the activation of energy production by Eiger signaling could also trigger another mechanism that decreases the tissue ATP level. It is possible that the tissue loses ATP simply because of the massive cell death caused by Eiger expression. Alternatively, the work by Temkin (2006) has shown that treatment with TNFα and the caspase inhibitor zVAD not only induces nonapoptotic cell death with ROS production but also decreases ATP because of the inhibition of adenine nucleotide translocase (ANT) by RIP1. A similar ANT-dependent inhibition mechanism could be involved in Eiger-induced cell death. Because neither RIP1 nor RIP3 has yet been identified in Drosophila, the mechanism by which the total ATP is regulated in Eiger-induced cell death remains to be elucidated (Kanda, 2011).
When scrib or dlg mutant cells are induced as clones in otherwise WT eye imaginal discs, most of these mutant cells are eliminated by Eiger–JNK-dependent cell death during development (Cordero, 2010; Igaki, 2009). This cell death could involve a caspase-independent mechanism, because the elimination of mutant cells is not fully suppressed by the overexpression of p35 compared with the blockage of JNK signaling. Thus, the mode of cell death triggered in scrib mutant clones is analogous to the mode of cell death triggered by the overexpression of Eiger in imaginal discs. These observations suggest that the regulation of energy production could be a crucial determinant of the susceptibility of tumor cells to cytotoxic stimuli (Kanda, 2011).
Interestingly, tumor cells frequently produce ATP by glycolysis in the cytosol rather than in the mitochondria, which is known as the Warburg effect. Because mitochondrial energy production generates cytotoxic ROS, cancer cells might increase their resistance to cytotoxic stimuli by reducing mitochondrial energy production. In this sense, mitochondrial energy production could act as a tumor suppressor. In fact, subunits of a TCA cycle enzyme, Succinate dehydrogenase (Sdh; SdhB, SdhC, and SdhD), are reported to be classical tumor suppressors in pheochromocytoma or paraganglioma. Furthermore, a specific isoform of pyruvate kinase, which is involved in glycolysis, is necessary for cellular metabolism to shift to aerobic glycolysis and the promotion of tumorigenesis. Similarly, the activity of pyruvate dehydrogenase (PDH), which links the glycolytic pathway to the TCA cycle by transforming pyruvate to acetyl-CoA, is suppressed in cancer cells, whereas the reactivation of PDH induces cell death in a solid tumor cell line and xenografts. Interestingly, this study found that the knockdown of Drosophila PDH (which is encoded by CG7010) strongly suppressed Eiger-induced cell death. Furthermore, the down-regulation of genes involved in glycolysis and the β-oxidation of fatty acids significantly suppressed the elimination of scrib cells from imaginal epithelia. These observations suggest that the regulation of cellular energy production or even the source of energy could be critical for controlling the susceptibility of cancer cells to cytotoxic stimuli such as TNFα (Kanda, 2011).
Highwire is an extremely large, evolutionarily conserved E3 ubiquitin ligase that negatively regulates synaptic growth at the Drosophila NMJ. Highwire has been proposed to restrain synaptic growth by downregulating a synaptogenic signal. This study identifies such a downstream signaling pathway. A screen for suppressors of the highwire synaptic overgrowth phenotype yielded mutations in wallenda, a MAP kinase kinase kinase (MAPKKK) homologous to vertebrate DLK and LZK. wallenda is both necessary for highwire synaptic overgrowth and sufficient to promote synaptic overgrowth, and synaptic levels of Wallenda protein are controlled by Highwire and ubiquitin hydrolases. highwire synaptic overgrowth requires the MAP kinase JNK and the transcription factor Fos. These results suggest that Highwire controls structural plasticity of the synapse by regulating gene expression through a MAP kinase signaling pathway. In addition to controlling synaptic growth, Highwire promotes synaptic function through a separate pathway that does not require Wallenda (Collins, 2006).
JNK signaling affects many cellular processes, often by regulating transcription factor activity that leads to changes in gene expression. A common downstream effector of JNK-mediated changes in gene expression is the AP-1 complex of Fos and Jun transcription factors, which can regulate synaptic growth at the Drosophila NMJ. To investigate whether Drosophila Fos or Jun (known as D-fos and D-jun, respectively) are required for highwire-dependent synaptic overgrowth, each was inhibited by expressing dominant-negative transgenes that contain the DNA binding and dimerization domains of Fos and Jun but lack the transcriptional activation domains. Expression of these dominant-negative transgenes in postmitotic neurons allowed circumvention of early embryonic requirements for D-fos and D-jun (Collins, 2006).
When FosDN and JunDN are neuronally expressed in a wild-type background, there is a modest trend toward inhibition of synaptic growth. When expressed in a highwire mutant background, the FosDN transgene confers dramatic suppression of the highwire synaptic phenotype, reducing bouton number and branching (42%) and increasing the intensity of staining for synaptic vesicle markers at the synapse. The reduction in highwire-dependent synaptic overgrowth is much greater than the reduction of growth in a wild-type background. In contrast, JunDN does not suppress the highwire phenotype. This suggests the existence of a pathway that is separate from AP-1, consistent with results in Drosophila demonstrating that D-Fos can act independently of D-Jun. The requirement for D-Fos in highwire synaptic overgrowth suggests that the highwire phenotype involves changes in gene expression rather than exclusively local changes to the synapse (Collins, 2006).
If FosDN acts downstream of Wallenda to inhibit synaptic overgrowth, it should also suppress the synaptic overgrowth caused by overexpressing wallenda. Indeed, when FosDN was coexpressed with UAS-wnd in neurons, FosDN could suppress the wallenda gain-of-function phenotype, leading to a 38% reduction in synaptic bouton number, a 52% reduction in synaptic branching, a 54% increase in bouton size, and a 3.8-fold increase in the intensity of staining of synaptic vesicle markers. This is consistent with D-Fos acting downstream of Wallenda to promote synaptic growth. Therefore, the synaptic overgrowth phenotypes caused by loss of highwire and by overexpression of wallenda are similar in their requirements for the transcription factor D-Fos (Collins, 2006).
Current models suggest that Highwire functions as an E3 ubiquitin ligase to downregulate a signaling pathway that promotes synaptic growth. This study identified a MAPKKK, Wallenda, whose protein levels are controlled by Highwire and the activity of ubiquitin hydrolases. Wallenda is both necessary for highwire-dependent synaptic overgrowth and sufficient to promote synaptic growth. Downstream of Wallenda, the MAP kinase JNK and transcription factor Fos are required for highwire-dependent synaptic overgrowth. It is proposed that Highwire restrains synaptic growth by downregulating the MAPKKK Wallenda, thereby inhibiting signaling through the JNK MAP kinase and the Fos transcription factor. In the absence of highwire, this signaling pathway is overactive, leading to changes in gene expression that result in excessive synaptic growth (Collins, 2006).
The regulation of the MAPKKK Wallenda is conserved in Drosophila and C. elegans (Nakata, 2005). In both organisms, the synaptic phenotype of highwire/rpm-1 requires the Wallenda/DLK-1 MAPKKK and downstream MAPK signaling. However, the downstream MAPK pathways diverge: in C. elegans, the rpm-1 phenotype requires a p38 MAP kinase (Nakata, 2005), while the highwire phenotype requires JNK signaling. This suggests that regulation of the specific MAPKKK Wallenda/DLK-1, rather than a particular downstream MAP kinase pathway, is a fundamental activity of Highwire and its orthologs (Collins, 2006).
Since Highwire functions as an E3 ubiquitin ligase to restrain synaptic growth, Wallenda is a compelling candidate target for the following reasons: (1) wallenda functions downstream of highwire and is essential for the synaptic overgrowth in highwire mutants; (2) increasing the levels of Wallenda by overexpression is sufficient to confer synaptic overgrowth; (3) Highwire regulates Wallenda protein levels through a posttranscriptional and most likely posttranslational mechanism. Each of the points above is conserved in C. elegans (Nakata, 2005 ). (4) Wallenda protein levels are regulated by ubiquitination in vivo, since inhibiting ubiquitination by overexpressing ubiquitin hydrolases increases the levels of Wallenda protein. (5) The RING domain of the C. elegans homolog rpm-1 can interact with the Wallenda homolog DLK-1 (Nakata, 2005) and stimulate its ubiquitination when both are overexpressed in 293T cells (Collins, 2006).
Targeting a MAPKKK, which sits at the top of a MAP kinase signaling pathway, is an attractive mechanism for spatially and temporally controlling a synaptogenic signal without affecting downstream components shared by multiple MAPK signaling cascades. Restraining MAP kinase signaling is essential for controlling diverse cellular processes, including cell proliferation, differentiation, and apoptosis. The targeting of MAPKKKs by specific ubiquitin ligases may be a powerful and general mechanism for regulating MAP kinase signals (Collins, 2006).
While Wallenda is an essential mediator of the highwire mutant phenotypes in both Drosophila and C. elegans, an endogenous synaptic function for Wallenda has not yet been identified in either organism: the wallenda mutants have surprisingly normal synapse morphology and function. This may be due to another pathway that compensates for the loss of wallenda function. Such redundancy would obscure the role of wallenda. A second possibility is that wallenda functions in an aspect of synaptic growth that is not detected or required under laboratory culture conditions. For instance, wallenda could promote synaptic growth as part of a structural plasticity program that responds to unknown experience-dependent stimuli. A third possibility is that Wallenda does not normally function at synapses, but its upregulation in highwire mutants causes a neomorphic phenotype. In this scenario, the regulation of Wallenda by Highwire is required for normal synaptic development, but endogenous Wallenda would not itself regulate the synapse. The neuropil and synaptic localization of Wallenda and the vertebrate homolog DLK (Hirai, 2005) is, however, consistent with a synaptic function (Collins, 2006).
As an activator of MAP kinase signaling, Wallenda and its homologs might also control other processes beyond the synapse. Functional studies in vertebrates suggest that DLK and JNK signaling regulate neuronal migration and axon outgrowth in the developing cortex (Hirai, 2002). Outside of the nervous system, DLK influences keratinocyte differentiation, and LZK is highly expressed in the pancreas, liver, and placenta. In Drosophila, wallenda mutants are female sterile. It is predicted that the regulation of DLK and LZK is conserved from worms and flies to vertebrates. Therefore, the vertebrate homologs of Highwire might regulate some of these neuronal and/or extraneuronal developmental processes (Collins, 2006 and references therein).
Highwire is a large, multidomain protein that, in addition to acting as an E3 ubiquitin ligase, has been shown to inhibit adenylate cyclase, influence TSC signaling and pteridine biosynthesis, and interact with the myc oncogene and the co-SMAD Medea. It is remarkable that throughout millions of years of evolution, members of the Highwire family have retained an exceptionally large size and complex domain structure. An attractive explanation for this conservation is that this molecule could serve as an intersection point for multiple signaling pathways, integrating MAP kinase and other signals during neural development (Collins, 2006).
The ubiquitin ligase activity alone could be responsible for regulating more than one downstream target. Interactions with components of TSC (tuberin/hamartin) and TGF-β signaling pathways suggest that Highwire might target either or both of these pathways. The model that Highwire regulates TGF-β signaling through interaction with the co-SMAD Medea has received considerable attention. Since the TGF-β pathway regulates synaptic growth at the NMJ, it has been proposed that synaptic overgrowth of highwire mutants is caused by overactivity of this pathway. Null alleles of wit, which completely disrupt TGF-β signaling at the NMJ, can partially suppress the highwire phenotypes: they partially suppress the increase in bouton number, but show little or no suppression of the reduced bouton size and the reduced intensity for synaptic vesicle markers. This partial suppression of highwire by wit is consistent with the model that overactive TGF-β signaling contributes to the highwire phenotype. However, the data are also consistent with the alternate model that TGF-β signaling and Highwire act in parallel pathways. An assay for the activity of TGF-β signaling is to stain for phosphorylated-MAD (phospho-MAD), the major transducer of BMP signals in Drosophila, in motoneuron nuclei. No change was detected in the levels of phospho-MAD staining in highwire mutants compared to wild-type. This assay is sensitive to changes in pathway activity—neuronal expression of the constitutively active type I receptor thick veins leads to a 40% increase in phospho-MAD staining. Interestingly, this increase in TGF-β signaling does not lead to excess synaptic growth. Combining a highwire mutant with expression of constitutively active thick veins does cause excess growth, but it does not lead to any further increase in phospho-MAD staining. These data are consistent with highwire and TGF-β signaling acting in parallel pathways (Collins, 2006).
Whether or not Highwire regulates TGF-β signaling, it is likely to target an additional pathway. Highwire not only restrains synaptic growth, but also promotes synaptic function. Synaptic function requires the ubiquitin ligase activity of Highwire and is sensitive to the levels of the ubiquitin hydrolase fat facets. This study demonstrates that this regulation of neurotransmitter release does not require Wallenda. Therefore, Highwire must regulate at least two distinct molecular pathways. If Wallenda is a substrate whose downregulation is essential for restraining synaptic growth, there is likely another substrate for Highwire whose downregulation promotes neurotransmitter release (Collins, 2006).
Downstream of Wallenda, the JNK MAP kinase and Fos transcription factor are required for the highwire synaptic morphology phenotype. Therefore, Highwire attenuates a JNK signaling pathway that presumably controls gene expression to regulate synaptic growth. Previous studies have implicated JNK-dependent transcriptional control in activity-dependent growth of the Drosophila NMJ. However, this previously described pathway is probably distinct from the JNK signal that is controlled by Highwire and activated by Wallenda. The previously described role for JNK requires AP-1, a heterodimer of Fos and Jun transcription factors; inhibiting either D-Fos or D-Jun disrupts this pathway. In contrast, highwire-induced overgrowth requires D-Fos, but not D-Jun. The Wallenda pathway could therefore involve a homodimer of D-Fos or another transcription factor that interacts with Fos. Such D-Jun-independent functions of D-Fos have been described previously in Drosophila. The differential requirement for transcription factors suggests that the output of Wallenda signaling cannot simply be activation of JNK, but instead activation of JNK in a particular spatial or temporal context, such as in the presence of cofactors that influence downstream signaling (Collins, 2006).
In addition to transcription factors, substrates for activated JNK include components of the cytoskeleton. Because the NMJ is distant from the motoneuron nucleus, and because vertebrate DLK colocalizes with tubulin in axonal regions of the brain, it was initially expected that the Highwire/Wallenda/JNK pathway would influence synaptic morphology through local action upon the synaptic cytoskeleton. Instead, a requirement was identified for a transcription factor and presumably changes in gene expression. However, this does not exclude an interaction with the cytoskeleton or local changes at the synapse. It is possible that Highwire regulates the Wallenda signal in the cell body. However, the observation that Wallenda accumulates in the synapse-rich neuropil and at the NMJ when Highwire is absent suggests that Wallenda could become activated at the synapse. This would imply the need for a mechanism to transport the activated JNK signal back to the nucleus. In addition, cell-wide changes in gene expression must then be translated into localized growth at the synapse. Activated Wallenda at the synapse is an attractive candidate to integrate changes in gene expression with regulation of the synaptic cytoskeleton to control synaptic growth (Collins, 2006).
Axon degeneration underlies many common neurological disorders, but the signaling pathways that orchestrate axon degeneration are unknown. This study found that dual leucine kinase (DLK) promotes degeneration of severed axons in Drosophila and mice, and that its target, c-Jun N-terminal kinase, promotes degeneration locally in axons as they committed to degenerate. This pathway also promotes degeneration after chemotherapy exposure and may be a component of a general axon self-destruction program (Miller, 2009).
Candidate components of axon breakdown pathways should be present in axons and activated by diverse cellular insults. One such candidate is DLK, a mitogen-activated protein kinase kinase kinase (MAP3K). One of DLK's downstream targets, the mitogen-activated protein kinase (MAPK) c-Jun N-terminal kinase (JNK), is activated following axonal injury. The hypothesis that DLK promotes axon degeneration using Drosophila olfactory receptor neuron (ORN) axotomy model. Freen fluorescent protein (GFP) was expresssed in ORNs to visualize their axons, which extend from cell bodies in the antennae into the antennal lobes of the brain and across a midline commissure. To severe ORN axons and induce degeneration, the antennae was removedfrom wildtype flies and mutants lacking the Drosophila ortholog of DLK, wallenda (wnd). Most wildtype axons degenerated within twenty-four hours, while wnd mutant axons were significantly preserved. Wnd is therefore required for normal axon degeneration in Drosophila (Miller, 2009).
Wnd could act within neurons to promote breakdown after injury or within surrounding cells to promote axon clearance. To distinguish between these possibilities, Wnd was expressed in the GFP-expressing subpopulation of ORNs in wnd mutant flies. Such Wnd expression was not sufficient to induce degeneration in the absence of injury. However, it was found that the Wnd expressing axons of these otherwise wnd mutant flies were not preserved twenty-four hours after axotomy. Thus, Wnd functions in an internal neuronal pathway that promotes injury-induced axon degeneration. Wnd may selectively promote injury induced axon degeneration, since no defects in the developmental pruning of mushroom body gamma-lobe axons (Miller, 2009).
To determine if DLK promotes Wallerian degeneration in mammals, dorsal root ganglion (DRG) cultures were used from littermate wildtype and DLK-deficient embryos. DRG axons were severed to induce degeneration and degeneration of the distal axon segment was evaluated. Twenty-four hours after severing, wildtype axons distal to the transection deteriorated into axon fragments, while DLK-deficient axons remained continuous. To quantify the extent of axon fragmentation, the fraction of total axonal area occupied by axon fragments (degeneration index, DI) was measured, and it was found that DLK-deficient axons were significantly preserved. This delay in fragmentation persisted for forty-eight hours. Since non-neuronal cells are eliminated in this DRG culture system, DLK must operate within mammalian neurons to promote axon breakdown. Neuronal DLK therefore promotes axon fragmentation after injury in flies and mice (Miller, 2009).
To determine if DLK promotes degeneration in response to multiple insults, the response was determined of DLK-deficient DRG axons to vincristine, a chemotherapeutic drug that induces axon degeneration in vitro, and whose dose-limiting side effects in patients include neuropathy. It was found that DLK-deficient axons were significantly protected from vincristine-induced fragmentation, suggesting that DLK operates in a general axon breakdown program (Miller, 2009).
To determine if disrupting DLK protects injured axons in vivo, the sciatic nerves of littermate wildtype and DLK-deficient adult mice were transected. Fifty-two hours post-transection, wildtype axons degenerated, whereas DLK-deficient axons were significantly preserved. Electron microscopy revealed that preserved axon profiles contain mitochondria and a cytoskeleton. Thus, normal Wallerian degeneration in vivo in adult mice requires DLK (Miller, 2009).
DLK is a MAP3K that can activate JNK and p38 via intermediary MAP2Ks. To determine whether either downstream kinase promotes axon degeneration, JNK and p38 were inhibited in the DRG axotomy model using wildtype cultures. Inhibition of JNK, but not p38, protected transected axons from fragmentation, and a significant delay in fragmentation persisted for over forty-eight hours. Thus JNK, like DLK, acts within neurons to promote axon degeneration (Miller, 2009).
Axon degeneration is hypothesized to comprise at least three distinct phases; (1) competence to degenerate, much of which is determined transcriptionally before axotomy, (2) commitment to degenerate, which occurs in the substantial delay period between injury and axon fragmentation, and (3) the execution phase, when axons fragment. If JNK';s primary role were to promote competence to degenerate, then JNK activity should be required prior to axotomy. This is not the case: applying the JNK inhibitor twenty-four hours prior to axotomy and then removing it just before axotomy was not protective. In contrast, JNK inhibition started concurrently with axotomy was protective. Thus, JNK promotes axon fragmentation after the competence period and acts within the severed distal axon segment (Miller, 2009).
JNK could commit axons to degenerate during the delay between injury and breakdown, or it could operate during the subsequent execution phase of axon breakdown. To test whether JNK activity is required during the execution phase, the JNK inhibitor was added three hours after axotomy, which is approximately nine hours before the onset of fragmentation. This treatment schedule spans the transition from the proposed commitment phase to the execution phase and the entire execution phase itself. Continuous JNK inhibition beginning three hours post-axotomy did not delay axon fragmentation. Therefore, JNK activity is not required during the execution phase of axon fragmentation. Rather, JNK activity is required during the early response to injury that commits the axon to breakdown hours later (Miller, 2009).
Converging lines of evidence suggest that there is a general internal axon self-destruction program, but its molecular components are unknown. This study shows that the MAP3K DLK and its downstream MAPK JNK are important elements of such a program. Disrupting this pathway delays axon fragmentation in response to both axotomy and the neurotoxic chemotherapeutic agent vincristine. Thus, a common self-destruction program may promote axon breakdown in response to diverse insults, and so may be targetable in multiple clinical settings (Miller, 2009).
Drosophila imaginal discs are monolayered epithelial invaginations that grow during larval stages and evert at metamorphosis to assemble the adult exoskeleton. They consist of columnar cells, forming the imaginal epithelium, as well as squamous cells, which constitute the peripodial epithelium and stalk (PS). A new morphogenetic/cellular mechanism for disc eversion has been uncovered. Imaginal discs evert by apposing their peripodial side to the larval epidermis and through the invasion of the larval epidermis by PS cells, which undergo a pseudo-epithelial-mesenchymal transition (PEMT). As a consequence, the PS/larval bilayer is perforated and the imaginal epithelia protrude, a process reminiscent of other developmental events, such as epithelial perforation in chordates. When eversion is completed, PS cells localize to the leading front, heading disc expansion. The JNK pathway is necessary for PS/larval cells apposition, the PEMT, and the motile activity of leading front cells (Pastor-Pareja, 2004).
One of the processes that best exemplify the dramatic changes that shape organisms is insect metamorphosis. In Drosophila and other holometabolous insects, most of the larval structures are replaced with new tissues that will give rise to the adult or imago. In particular, the adult epidermis with the exception of the abdominal structures develops from imaginal epithelial discs. During larval stages, the primordia of imaginal discs, set during embryogenesis, invaginate and grow to become flattened sacs arranged in a monolayer epithelium connected to the larval epidermis by a stalk. The mature discs contain two populations of cells, a columnar epithelium that will give rise to most of the adult structures and a thinner and more squamous peripodial epithelium (PE) with a reduced contribution to adult tissues. Upon metamorphosis, the imaginal discs undergo striking morphological changes, everting, expanding, and fusing to ipsilateral and contralateral adjacent discs generating the adult exoskeleton (Pastor-Pareja, 2004).
The process of movement and sealing of imaginal discs and, in general, epithelial sheets can be subdivided into three sequential steps: (1) leading cells are specified and brought into position; (2) cells execute coordinated forward movements by changing shape and/or migrating over a substratum, and (3) sheets merge and fuse. Most recent work on disc morphogenesis has focused on the cellular and molecular events underlying their late expansion and fusion, while the mechanisms involved in disc eversion have been poorly explored in vivo (Pastor-Pareja, 2004).
In late third instar larvae, the steroid molting hormone 20-hydroxyecdysone is believed to coordinate the almost simultaneous eversion of all discs by inducing a contraction of the PE. This is thought to drive movement of the appendages to the outside of the larval epidermis through relaxed and widened disc stalks. This classical view is supported by in vitro studies showing that treatment of cultured discs with ecdysone is sufficient to induce eversion and that contraction of an intact PE is necessary to achieve this goal. These descriptive reports have led to the proposition that cell shape changes (longitudinal contraction in the PE and circumferential elongation at the disc stalks) are sufficient for imaginal disc eversion. However, there are as yet no data to confirm that this mechanism exists in vivo and no convincing explanation on how a stalk of no more than ten cells in diameter could achieve the width required to allow the entire disc (more than 60,000 cells) to pass through. Further, this accepted view neglects earlier proposals suggesting a different eversion mechanism mediated by the rupture of the PE. A model supported by fate maps has been developed for the PE of Calliphora, a related dipteran, imaginal wing discs (Pastor-Pareja, 2004).
Several studies have revealed a requirement for cytoskeletal components and a number of signal transduction molecules for imaginal disc morphogenesis during the first hours of metamorphosis. The latter include the Drosophila AP-1 transcription factors, D-Jun and D-Fos (Kayak [Kay]), and an upstream kinase cascade homologous to the Jun-NH2-terminal kinase (JNK) pathway in mammals. The core of this cascade is formed by the stress-activated kinases JNKK and JNK. In Drosophila, JNKK and JNK homologs are encoded by the genes hemipterous (hep) and basket (bsk). JNK signaling mutant larvae do not spread their discs in the process of thorax closure. This phenotype is accompanied by a loss of puckered (puc) expression in the disc stalk and the PE. Puc is a dual-specificity phosphatase that selectively inactivates Bsk and, thus, is thought to act in a negative feedback loop. JNK activity is necessary to maintain the adhesion of the imaginal leading edge cells to their larval substrate and to promote actin dynamics (lamellipodia and filopodia formation). It has been shown that this signaling cascade also regulates the process of embryonic dorsal closure, where the embryonic epidermis fuses along the dorsal midline. Based on these similarities, it has been suggested that a conserved mechanism regulates the spreading and fusion of epithelial sheets (Pastor-Pareja, 2004).
A new morphogenetic/cellular mechanism has been uncovered for disc eversion based on histological sections and direct observation of imaginal morphogenesis in vivo. At the onset of metamorphosis, imaginal discs coordinately appose their peripodial sides and stalks (PS cells) to the larval epidermis. Then, eversion proceeds through the progressive invasion of the larval epidermis by PS cells undergoing a pseudo-epithelial-mesenchymal transition (PEMT). Multiple perforations in the peripodial/larval bilayer are thus generated: these coalesce with the disc stalk into a single hole, widening the gap and allowing disc evagination. When eversion is complete, the PS cells localize to the leading front of the discs, spearheading their expansion over larval cells. The roles of the JNK pathway at discrete steps of disc morphogenesis progression have been analyzed. The JNK cascade functions to promote the apposition of PS and larval cells, to determine the degree of PEMT and the motility of leading edge/PS cells, and to maintain the adhesion between the larval and imaginal tissue. It is proposed that this molecular mechanism can be relevant to morphogenetic processes of perforation of transient epithelia in different phyla (Pastor-Pareja, 2004).
The current view of imaginal disc eversion asserts that the externalization of appendage primordia proceed through widened discs' stalks during early pupal development. However, a detailed analysis of PS cell markers appears to challenge this simple inversion mechanism (Pastor-Pareja, 2004).
In early third instar imaginal wing discs, the gene puc is expressed at high levels in stalk cells and some PE cells. This expression evolves through the third instar until all PS cells (about 700 in the mature wing disc) express puc at the white prepupa stage (0-1 hr hours after puparium formation [APF]). These dynamic changes of puc expression are also observed in leg, haltere, and eye discs. The PS expression of puc strictly depends on JNK activity, and it is abolished from mutant hep (JNKK) larvae or after Puc overexpression. Thus, a JNK signaling feedback loop, first described during embryonic dorsal closure, is shared by PS cells at the onset of the eversion and closure of the discs. During wing disc eversion, only cells found at the edge of the hole through which the disc everts and at the leading front mediating fusion to adjacent prothoracic, mesothoracic, and metathoracic discs show puc expression, and hence JNK activity. Importantly, marking all cells that have expressed puc as well as their descendants shows that puc-expressing cells do not change their identity, nor do they die or get excluded from the epithelium until the end of the disc fusion process, when most of these cells are lost. Hence, all PS cells are recruited to the front edge during disc eversion (Pastor-Pareja, 2004).
These findings lead to a topological dilemma. In order to reach their final position at the leading front, the PS cells would need to reposition themselves within the epithelia. Although this rearrangement just could be achieved through a massive constriction of the PE, a complementary mechanism has been uncovered, which involves larval epidermis perforation and PE cells intercalation (Pastor-Pareja, 2004).
At third instar larval stages, the wing disc obliquely hangs from the larval epidermis, which is separated from the peripodial surface of the disc at the notum level by several larval muscles and tracheal tubules. The disc and the larval epidermis are isolated by their corresponding extracellular basal lamina. During late third instar stages and the first hours APF, the notum-wing side of the disc folds progressively to acquire the adult organ shape. At the initiation of pupariation, the disc affixes to the larval epidermis through its peripodial side. At 3 hr APF, the PS cells lose their squamous shape to adopt a more rounded one and are found in close contact with the larval epidermal cells via their basal surfaces. Multiple actin-rich protrusions lead this apposition. At this step, the basal lamina in between both layers degrades, leading to an intimate adhesion (Pastor-Pareja, 2004).
Once imaginal discs appose the larval epidermis, PS cells, mostly around the disc stalk, invade the larval epithelium, gradually replacing the larval cells at the pupal surface without compromising the integrity of the peripodial sheet. Several holes are opened in the peripodial/larval bilayer, which within a few minutes converge with the original stalk into a single aperture. Interfering with apoptosis by overexpressing the P35 cell death inhibitor in imaginal and larval tissues does not affect epithelial perforation and disc eversion (Pastor-Pareja, 2004).
Following coalescence, the progressive widening of the hole by intercalation of PS cells at the leading front was observed. A cell lineage analysis was performed and multiple clones of PS cells were found, that remain compact up to the third instar larval stage in the PE and lose cohesion during eversion. Thus, PS cells appear to change neighbors, become extremely active, and emit and retract filopodia and lamellipodia at their front and rear ends. They squeeze in between themselves and the rest of the epithelium (planar intercalation), migrating to and expanding the front of the disc, and leading the migration over the larval tissue (Pastor-Pareja, 2004).
Simultaneous to wing disc eversion, all legs and haltere discs evert using the same mechanism (Pastor-Pareja, 2004).
One hallmark of epithelial cells is their distinct apico-basal cell polarity. This polarity depends on a set of intercellular connections, which encircle epithelial cells at the border of the apical and basal-lateral membrane domains. The cells in insect epithelial tissues are interconnected by zonula adherens (ZAs), which function in both cellular adhesion and signaling. DE-cadherin is the major constituent of the ZAs in a complex with Armadillo (Arm, ß-catenin) and Dalpha-catenin. In addition, epithelia of flies and other invertebrates exhibit septate junctions, which are located basally to the ZAs. Septate junctions prevent diffusion through the pericellular space and are functionally equivalent to vertebrate tight junctions (Pastor-Pareja, 2004).
All imaginal disc cells at the third instar larval stage presented ZAs in an apical belt. During disc eversion, however, it was found that ZAs components delocalize from the free edges of the PS cells, remaining cytoplasmic at the edges of the perforations arising through the PS/larval bilayer and in those PS cells leading the spreading of the discs over the larval tissues. As a consequence, ZAs are lost in these cells . Moreover, septate junction components, such as Coracle and Disc Large are also found to be missing from the membranes of leading front cells (Pastor-Pareja, 2004).
The loss of apico/basal polarity and adhesion of the PS cells during disc eversion is reminiscent of an epithelial-mesenchymal transition (EMT), as described for mesoderm and neural crest cells in vertebrates, and for the acquisition of the invasive phenotype in carcinomas (Pastor-Pareja, 2004).
The JNK signaling cascade dictates the expression of puc in all PS cells but their early specification appears not to be affected by lowering the level of JNK activity, since the complete absence of Hep function did not alter either their number or morphology in third instar larval discs. However, several mutant phenotypes have provided strong evidence for a leading role of the JNK pathway in imaginal disc fusion and disc eversion; e.g., hep mutants occasionally show uneverted wing discs lying inside the body of the pupa. When and how is JNK signaling needed? Transversal semi-thin sections, at 6 ± 1 hr APF, long after closure is completed in wild-type, of hepr75 (a strong hypomorphic mutation) pupae show a range of phenotypic defects (classes I to III). Class I corresponds to a complete failure of PS/larval apposition (40% of individuals); in class II, discs apposed to the larval tissue but did not complete their eversion (50%); the mildest condition, class III, refers to discs that everted completely and advanced to some extent but were unable to fuse (10%). By the complete inactivation of the JNK signaling cascade through the ubiquitous overexpression of puc (from 48-60 hr before puparium formation onward), a fully penetrant failure was found of disc apposition to the larval epidermis (class I phenotype). A delayed or reduced (in a puc heterozygous background) overexpression of puc produced less severe class II and III phenotypes. Thus, the JNK cascade appears to be essential for PS and larval cell apposition and, as suggested by the observed phenotypic progression, may also be involved in later steps during eversion (Pastor-Pareja, 2004).
JNK activity levels also affect the degree of PEMT in PS cells. ZAs are absent from leading front cells and the membrane localization of DE-cadherin and Arm is progressively lost, as PS cells moved closer to the free edge. However, in hepr75 mutant pupae (class III), the cells at the leading front of the disc do not delocalize either Arm or DE-cadherin in the free edge, suggesting that partial loss of JNK signaling blocks the correct transition of these cells from immotile epithelial to migrating and invading leading front cells. Further, a surplus of JNK activity in PS cells in pucE69F-GAL4 mutants conveyed the transition of an excess of PS cells to a mesenchymatic phenotype. Hence, an adequate balance of JNK activity is key to control the level of PEMT. Too few mesenchymal-like PS cells restrain the ability of discs to evert and spread, while too many transformed cells affect the ability of discs to appropriately fuse. Further, pucE69F-GAL4 mutants also showed enhanced cell motility and massive cell detachment from the free edges of the epithelium. These cells adopted a rounded shape but remained in close proximity, establishing transient contacts. Conversely, leading cells in hepr75 mutants do not show any migratory activity. Hence, the JNK pathway regulates not only the adhesive properties of PS cells, but also their motility (Pastor-Pareja, 2004).
In summary, the JNK signaling cascade participates in four key steps in the process of disc eversion: (1) the expression of puc in PS cells; (2) the apposition of PS and larval cells; (3) the regulation of the adhesive and motile properties of PS cells as they undergo PEMT, and (4) the maintenance of the adhesion between the larval and imaginal tissue (Pastor-Pareja, 2004).
Thus, within the first 5 hr after puparium formation, the precursors of the adult structures evert. Multiple evidences show that eversion is mediated by actin microfilaments contraction, which modulate a general change of morphology of PS and larval cells driving the inside-out eversion of the disc. Several observations, however, suggest that other morphogenetic mechanisms are also involved (Pastor-Pareja, 2004).
(1) An imaginal disc is a rigidly determined primordium, which allows the construction of fate maps. Surprisingly, peripodial fate maps of Calliphora, a related diptera, show that adjacent territories develop into nonadjacent adult pleural structures, suggesting that the peripodial layer splits during metamorphosis (Pastor-Pareja, 2004).
(2) PS cells expressing puc relocate during eversion to the leading front. Thus, intercalation of PS cells appears to be concurrent to eversion (Pastor-Pareja, 2004).
(3) Pupal serial sectioning shows that, at eversion, imaginal discs appose to the larval epidermis through their peripodial side. Just before eversion, PS cells lose their basal lamina and detach from the extracellular matrix (Pastor-Pareja, 2004).
(4) Preceding disc eversion, in vivo time-lapse reveals the opening of larval/peripodial gaps, which are the outcome of the invasive behavior and planar intercalation (PEMT) of PS cells (Pastor-Pareja, 2004).
In summary, the evagination of imaginal disc can be divided into the following sequential steps: (1) an overall positional change of the imaginal discs leading to the confrontation and apposition of the PS and the larval epidermis; (2) a regulated modulation (PEMT) of PS cells, which involves the downregulation of their cell-cell adhesion systems and allows them to move into their local neighborhood and invade the larval epithelium; (3) the fenestration of the peripodial/larval bilayer and the formation of an unbound peripodial leading front, which will direct imaginal spreading by planar cell intercalation, and (4) a bulging of the imaginal tissue (Pastor-Pareja, 2004).
Once the hole is opened, the planar intercalation of PS cells ensures that, first in the hole and later in the leading front, all four dorsal, ventral, anterior, and posterior compartments of the wing disc are represented. This mechanism also guarantees the maintenance of a continuous epithelial barrier (Pastor-Pareja, 2004).
The PcG proteins function through cis-regulatory elements called PcG response elements (PREs), which enable them to bind and to maintain the state of transcriptional silencing over many cell divisions. PcG proteins operate in two key evolutionarily conserved chromatin complexes, and reduced expression of these complexes, as found in PcG mutants, results in the derepression of PRE-controlled genes. To determine whether PcG silencing is modulated in regenerating tissue, the FLW-1 line, which contains a lacZ reporter gene under the control of the Fab7 PRE, was used. Prothoracic leg discs silent for lacZ expression were fragmented and transplanted into the abdomen of host flies. Flies were fed with 5-bromodeoxyuridine (BrdU) to mark the regenerated tissue (the blastema). In uncut discs, there was little proliferation and expression of lacZ was undetectable. On fragmentation, however, lacZ was expressed in the blastema. To confirm that this derepression was due to a reduction in PcG silencing and not simply to massive proliferation at the wound site, the line LW-1 was used; this line lacks the Fab7 PRE and is normally silent, but it can be activated by induction of GAL4. Neither uncut nor cut leg discs of the LW-1 line showed expression of lacZ after transplantation (Lee, 2005).
To show that transdetermination takes place only in cells with downregulated PcG function, fragmented leg discs of the FLW-1 line were stained for lacZ expression and for Vg in order to visualize the transdetermination to wing fate. It was consistently observed that the Vg staining lay within the lacZ expression domain, suggesting that PcG genes are downregulated in the blastema, enabling PRE-silenced genes to be reactivated according to new morphogenetic cues (Lee, 2005).
To investigate direct targets of PcG regulation that, when reactivated, might contribute to transdetermination, the PREs predicted at the wg and vg genes were tested and both were found to be controlled by PcG proteins. The fact that both the transgenic vg-lacZ reporter construct (which lacks the PRE) and the endogenous vg gene were upregulated in the blastema suggests that PcG proteins may affect vg expression both indirectly (for example, through wg) and directly by means of the vg PRE (Lee, 2005).
JNK signalling in Drosophila is crucial for wound healing and is implicated in many different developmental processes, such as dorsal and thorax closure. hemipterous encodes the JNK kinase (JNKK) that activates the Drosophila JNK Basket. Products of DJun and kayak (the Drosophila homologue of Fos) form the AP-1 transcription factor. A downstream target of JNK signalling is puckered (puc), which encodes a phosphatase that selectively inactivates Basket and thus functions in a negative feedback loop. The expression of puc thus mirrors JNK activity. Because wound healing takes place after fragmentation, it was reasoned that activation of the JNK pathway might be causing the downregulation of PcG proteins in the blastema. The pucE69 line, which carries a P(lacZ) insertion at the puc locus, was used to monitor JNK activity. During the third-instar larval stage puc is not expressed and thus JNK signalling was not activated in leg discs. As expected, however, puc was expressed on fragmentation in all cells at the annealing cut edge (Lee, 2005).
To check whether cells that have activated the JNK pathway also show transdetermination, fragmented leg discs of flies carrying the puc-lacZ reporter and vgBE-Gal4; UAS-GFP constructs were transplanted. In these flies, cells that adopted a wing fate were identified by their expression of green fluorescent protein (GFP). Two days after fragmentation, weak residual puc-lacZ staining was still visible in the central region of the disc. puc-lacZ staining is known to decline rapidly after wound healing is completed. It was found that stronger staining was visible along the cut site, probably owing to ongoing wound healing. On comparison of puc-lacZ staining and GFP fluorescence, JNK-active cells showed a substantial overlap with transdetermined cells; thus, it is concluded that JNK signalling is activated in cells that undergo transdetermination (Lee, 2005).
JNK signalling affects the transcription of numerous genes, including those encoding chromatin regulating factors. Therefore whether JNK signalling can downregulate the PcG proteins required for transdetermination was examined. A constitutively active form of hep was overexpressed in UAS-hepact; hsGal4 flies by a heat-shock pulse. Activating the JNK pathway caused a downregulation of some PcG genes, such as Pc, ph-p and E(Pc). No downregulation of these genes was observed in wild-type larvae before and after heat shock, indicating that this was not an unspecific heat-shock response. Expression was examined of two genes of the Trithorax group (ash1 and brm) that function antagonistically to PcG proteins, but found no upregulation on JNK induction (Lee, 2005).
To show further that JNK has a specific effect on PcG proteins, the analogous experiment was carried out in mammalian cells. The JNK pathway can be activated in mouse embryonic fibroblasts by exposing the cells to ultraviolet light. The expression of MPh2 (mouse polyhomeotic2) was examined because this mammalian PcG gene is expressed in these cells. The expression of MPh2 was decreased on JNK induction, but after treatment with a specific JNK inhibitor it was partially restored. In addition, to show that the downregulation of PcG genes is directly controlled by AP-1, chromatin immunoprecipitation was carried out using antibodies against Fos on chromatin from UAS-hepact; hsG4 and kay1 mutant flies. Enrichment of Fos on the promoter region of ph-p was observed, but no enrichment in chromatin from flies lacking Fos. This finding suggests that AP-1 binds directly to this region to regulate negatively the transcription of ph-p (Lee, 2005).
If activation of JNK signalling in the blastema indeed leads to a downregulation of PcG genes, then impairment of the JNK pathway should result in reduced efficiency of transdetermination. The transdetermination behaviour of wild-type discs was compared with that of discs bearing mutations in the JNKK hep. The transdetermination events were classified into three categories: large regions, small regions, and no regions of transdetermination. In wild-type discs only large regions were detected. In males hemizygous for hep1 (a weak hypomorphic allele), most transplanted leg discs had large transdetermined regions; however, a substantial proportion showed only small regions of transdetermination and a few showed no transdetermination event. In flies heterozygous for hepr75 (a null allele which is hemizygous lethal), most discs showed no or only small regions of transdetermination, and large regions were rarely seen. The morphology of the regenerated discs seemed unaffected in these mutants, indicating that the decline of transdetermination efficiency was not due to inefficient wound healing (Lee, 2005).
This study has shown that PcG genes are downregulated by JNK signalling. Because many developmental regulators need to be switched, the role of PcG downregulation may be to render the cells susceptible to a change in cell identity by shifting the chromatin to a reprogrammable state. Transdetermination has been ascribed to the action of ectopic morphogens, which induce cells to activate incorrect gene cascades. Without doubt, wg and decapentaplegic signalling must be crucially involved in this process, because transdetermination does not result from any random cut but occurs preferentially when cuts are made through particular regions of the disc called 'weak points', which are regions of high morphogen. Inappropriate or overextreme downregulation of the PcG system by JNK in sensitive cells of the weak points thus may create such aberrant local patterns. Indeed, the data indicate that at least the two patterning genes, wg and vg, may be direct targets of the PcG. Notably, hyperactive Wnt signalling can also induce a switch in lineage commitment in mammals, implying that signalling pathways are a potent inducer of cell fate changes in many organisms (Lee, 2005).
Another study has shown that regenerating and transdetermining cells in the blastema have a distinct cell-cycle profile in contrast to the surrounding normal disc cells. It has been proposed that this change in cell-cycle regulation is a prerequisite for the change in cell fate. Indeed, PcG targets include genes involved in cell-cycle regulation, suggesting that this initial step is part of the complete reprogramming cascade required for the regenerating cells to achieve multipotency. Downregulation of PcG silencing by JNK seems to be a fundamental, evolutionarily conserved mechanism of cell fate change and thus may also have implications for studies of stem cell plasticity and tissue remodelling (Lee, 2005).
The precise number and pattern of axonal connections generated during brain development regulates animal behavior. Therefore, understanding how developmental signals interact to regulate axonal extension and retraction to achieve precise neuronal connectivity is a fundamental goal of neurobiology. This question was investigated in the developing adult brain of Drosophila. Extension and retraction is regulated by crosstalk between Wnt, fibroblast growth factor (FGF) receptor, and Jun N-terminal kinase (JNK) signaling, but independent of neuronal activity. The Rac1 GTPase integrates a Wnt-Frizzled-Disheveled axon-stabilizing signal and a Branchless (FGF)-Breathless (FGF receptor) axon-retracting signal to modulate JNK activity. JNK activity is necessary and sufficient for axon extension, whereas the antagonistic Wnt and FGF signals act to balance the extension and retraction required for the generation of the precise wiring pattern (Srahna, 2006).
Based on the observation that blocking Fz2 results in decreased numbers of dorsal cluster neuron (DCN) axons in the medulla, it was reasoned that Fz2 could be a receptor for a putative stabilization signal. Since Fz2 and Fz are partially redundant receptors for the canonical Wnt signaling pathway, expression of the canonical Wnt ligand Wingless (Wg) was investigated in the brain during pupation. However, no Wg expression was detected in the pupal optic lobes, suggesting that Wg is unlikely to be involved in regulating DCN axon extension. Therefore, the expression of Wnt5, which has been shown to be involved in axon repulsion and fasciculation in the embryonic CNS, was investigated. Anti-Wnt5 staining revealed widely distributed Wnt5 expression domains beginning at PF and lasting throughout pupal development and into adult life. Wnt5 is strongly expressed in the distal medulla and is also present on axonal bundles crossing the second optic chiasm.The number of DCN axons crossing to the medulla was examined in wnt5 mutant flies. The number of DCN axons crossing the optic chiasm is reduced from 11.7 to 7.9 in the absence of wnt5, suggesting that it may play a role in stabilizing DCN axons (Srahna, 2006).
Next, the requirement of the Wnt signaling adaptor protein Dsh was tested. In animals heterozygous for dsh6, a null allele of dsh, the average number of DCN axons crossing between the lobula and the medulla is reduced from 11.7 to 7.6 with 78.5% showing less than eight axons crossing. Signaling through Dsh is mediated by one of two domains. Signaling via the DIX (Disheveled and Axin) domain is thought to result in the activation of Armadillo/β-Catenin. DEP (Disheveled, Egl-10, Pleckstrin) domain-dependent signaling results in activation of the JNK signaling pathway by regulation of Rho family GTPase proteins during, for example, convergent extension movements in vertebrates. To uncover which of these two pathways is required for DCN axon extension the dsh1 mutant, deficient only in the activity of the DEP domain, was tested. Indeed, in brains from dsh1 heterozygous animals the number of extending axons was reduced from 11.7 to 7.4. In flies homozygous for the dsh1 allele the average number of axons crossing was further reduced to 4.7, with all the samples having less than six axons crossing. In contrast, the DCN-specific expression of Axin, a physiological inhibitor of the Wnt canonical pathway, did not affect the extension of DCN axons. Similarly, expression of a constitutively active form of the fly β-Catenin Armadillo also had no apparent effect on DCN extension. Finally, whether Wnt5 and Dsh interact synergistically was tested. To this end, wnt5, dsh1 trans-heterozygous animals were generated. These flies show the same phenotype as flies homozygous for dsh1, suggesting that Wnt5 signals through the Dsh DEP domain (Srahna, 2006).
To determine if dsh is expressed at times and places suggested by its genetic requirement in DCN axon outgrowth, the distribution of Dsh protein during brain development was examined. Dsh protein is ubiquitously expressed during brain development. High expression of Dsh is detected in the distal ends of DCN axons at about 15% PF shortly before they extend across the optic chiasm toward the medulla. In general, higher levels of Dsh were observed in the neuropil than in cell bodies (Srahna, 2006).
In summary, these data indicate that the stabilization of DCN axons is dependent on the Dsh protein acting non-canonically via its DEP domain. Importantly, the axons that do cross in dsh mutant brains do so along the correct paths. This suggests that, like JNK signaling, Wnt signaling regulates extension, but not guidance, of the DCN axons (Srahna, 2006).
Wnt signaling to Dsh requires the Fz receptors. To examine if the effect of Wnt5 on DCN axon extension is also mediated by Fz receptors, the number of DCN axons crossing the optic chiasm in was counted fz, fz2, and fz3 mutants. There was no significant change in the number of axons crossing in the brain of fz3 homozygous animals. In contrast, in brains heterozygous for fz and fz2, the number of the axons crossing was reduced from 11.7 to 6.6 (fz) and 6.9 (fz2), with 71% and 85.7%, respectively, showing less than eight axons crossing. These data suggest that DCN axons respond to Wnt5 using the Fz and Fz2 receptors, but not Fz3. To determine whether the Fz receptors act cell-autonomously in individual DCNs, single-cell clones doubly mutant for fz and fz2 were generated and the number of DCN axons crossing the optic chiasm was counted. In contrast to wild-type cells, where 37% of all DCN axons cross, none of the fz, fz2 mutant axons reach the medulla. To test whether wnt5, fz, and fz2 genetically interact in DCNs, flies trans-heterozygous for wnt5 and both receptors were examined. Flies heterozygous for both wnt5 and fz mutations show a strong synergistic loss of DCN axons (11.7 to 3.7) and in fact have a phenotype very similar to that of flies homozygous for dsh1. Flies doubly heterozygous for wnt5 and fz2 also show a significant decrease in DCN axons (5.7), compared with either wnt5 (~8) or fz2 (8.5) mutants. These data indicate that the genetic interaction between wnt5 and fz is stronger than the interaction between wnt5 and fz2 (Srahna, 2006).
Examination of the expression domains of Fz and Fz2 in the developing brain supports the possibility that they play roles in stabilizing DCN axons. Both Fz and Fz2 are widely expressed in the developing adult brain neuropil. In addition, Fz is expressed at higher levels in DCN cell bodies (Srahna, 2006).
The observation that the wnt5 null phenotype can be enhanced by reduction of Fz, Fz2, or Dsh suggests that another Wnt may be partially compensating for the loss of Wnt5. To test this possibility, flies heterozygous for either wnt2 or wnt4 were examined. wnt2 heterozygotes display a reduction of DCN axon crossing from 11.7 to 7.3, whereas no phenotype was observed for wnt4. Thus, wnt2 and wnt5 may act together to stabilize the subset of DCN axons that do not retract during development. In summary, these results support the model that Wnt signaling via the Fz receptors transmits a non-canonical signal through Dsh resulting in the stabilization of a subset of DCN axons (Srahna, 2006).
Data is provided that supports the hypothesis that the regulation of JNK by Rac1 modulates DCN axon extension. As such attempts were made to determine how Wnt signaling might interact with Rac1 and JNK. The opposite phenotypes of dsh and Rac1 loss-of-function suggest that they might act antagonistically. To determine if Rac1 is acting upstream of, downstream of, or in parallel to Dsh in DCN axon extension, dominant-negative Rac1 was expressed in dsh1 mutant flies. If Rac1 acts upstream of Dsh, the dsh1 phenotype (i.e., decreased numbers of axons crossing the optic chiasm) is expected. If Rac1 acts downstream of Dsh, the Rac1 mutant phenotype (i.e., increased number of axons crossing) would be expected If they act in parallel, an intermediate, relatively normal phenotype is expected. Increased numbers of axon crossing were observed, suggesting that Rac1 acts downstream of Dsh during DCN axon extension and that Dsh may repress Rac1 (Srahna, 2006).
Next, whether Dsh control of DCN axon extension is mediated by the JNK signaling pathway acting downstream of Wnt signaling was tested, as the similarity of their phenotypes suggests. If this were the case, activating JNK signaling should suppress the reduction in Dsh levels. Conversely, reducing both should show a synergistic effect. Therefore the JNKK hep was expressed in dsh1 heterozygous flies and it was found that the hep gain-of-function is epistatic to dsh loss-of-function. Furthermore, reducing JNK activity by one copy of BSK-DN in dsh1 mutant animals results in a synergistic reduction of extension to an average of 0.8 axons with 60% showing no axons crossing and no samples with more than three axons. In summary, the results of genetic analyses suggest that Wnt signaling via Dsh enhances JNK activity through the suppression of Rac1 (Srahna, 2006).
Dsh appears to promote JNK signaling and to be expressed in DCN axons prior to their extension toward the medulla early in pupal development. Since JNK signaling is required for this initial extension, it may be that Dsh also plays a role in the early extension of DCN axons. To test this possibility, DCN axon extension was examined at 30% pupal development in dsh1 mutant brains. In wild-type pupae, essentially all (~40) DCN axons extend toward the medulla. In contrast, in dsh1 mutant pupae, a strong reduction in the number of DCN axons crossing the optic chiasm between the lobula and the medulla was observed (Srahna, 2006).
Although the genetic data indicate that Dsh- and Rac-mediated signaling have sensitive and antagonistic effects on the JNK pathway, they do not establish whether the Dsh-Rac interaction modulates JNK's intrinsic activity. To test this, the amount of phosphorylated JNK relative to total JNK levels in fly brains was evaluated by Western blot analysis using phospho-JNK (P-JNK) and pan-JNK specific antibodies. Then it was determined if Dsh is indeed required for increased levels of JNK phosphorylation. Dsh1 mutant brains showed a 25% reduction in P-JNK consistent with a stimulatory role for Dsh on JNK signaling. The reduction caused by loss of Dsh function is reversed, when the amount of Rac is reduced by half, consistent with a negative effect of Rac on JNK signaling downstream of Dsh. These data support the conclusion that Dsh and Rac interact to regulate JNK signaling by modulating the phosphorylated active pool of JNK (Srahna, 2006).
Taken together, these data suggest that during brain development DCN axons extend under the influence of JNK signaling. A non-canonical Wnt signal acting via Fz and Dsh ensures that JNK signaling remains active by attenuating Rac activity. In contrast, activation of the FGFR activates Rac1 and suppresses JNK signaling. These data support a model whereby the balance of the Wnt and FGF signals is responsible for determining the number of DCN axons that stably cross the optic chiasm. To test this model, FGFR levels were reduced, using the dominant-negative btl transgene, in dsh1 heterozygous flies. It was found that simultaneous reduction of FGF and Wnt signaling restored the number of axons crossing the optic chiasm to almost wild-type levels (10.2, with 33% of the samples indistinguishable from wild-type, suggesting that the two signals in parallel, act to control the patterning of DCN axon connectivity (Srahna, 2006).
These data suggest the following model of DCN axon extension and retraction. DCN axons extend due to active JNK signal. These axons encounter Wnt5 and probably Wnt2 as well, resulting in activation of Disheveled. Disheveled, via its DEP domain, has a negative effect on the activity of the Rac GTPase, thus keeping JNK signaling active. After DCN axons cross the second optic chiasm they encounter a spatially regulated FGF/Branchless signal that activates the FGFR/Breathless pathway. Breathless in turn activates Rac, which inhibits JNK signaling in a subset of axons. These axons then retract back toward the lobula. The wide expression of the different components of these pathways and the modulation of JNK phosphorylation by Dsh and Rac in whole-head extracts strongly suggests that this model may apply to many neuronal types (Srahna, 2006).
Cells in intestinal epithelia turn over rapidly due to damage from digestion and toxins produced by the enteric microbiota. Gut homeostasis is maintained by intestinal stem cells (ISCs) that divide to replenish the intestinal epithelium, but little is known about how ISC division and differentiation are coordinated with epithelial cell loss. This study shows that when enterocytes (ECs) in the Drosophila midgut are subjected to apoptosis, enteric infection, or JNK-mediated stress signaling, they produce cytokines (Upd, Upd2, and Upd3) that activate Jak/Stat signaling in ISCs, promoting their rapid division. Upd/Jak/Stat activity also promotes progenitor cell differentiation, in part by stimulating Delta/Notch signaling, and is required for differentiation in both normal and regenerating midguts. Hence, cytokine-mediated feedback enables stem cells to replace spent progeny as they are lost, thereby establishing gut homeostasis (Jiang, 2009).
Rates of cell turnover in the intestine are likely to be in constant flux in response to varying stress from digestive acids and enzymes, chemical
and mechanical damage, and toxins produced by both commensal and
infectious enteric microbiota. This study shows feedback from
differentiated cells in the gut epithelium to stem and progenitor cells
is a key feature of this system. Genetically directed enterocyte
ablation, JNK-mediated stress signaling, or enteric infection with P. entomophila all disrupt the Drosophila
midgut epithelium and induce compensatory ISC division and
differentiation, allowing a compromised intestine to rapidly
regenerate. Other recent reports note a similar regenerative response
following three additional types of stress: detergent (DSS)-induced
damage (Amcheslavsky, 2009), oxidative stress by paraquat (Biteau, 2008), and enteric infection with another less pathogenic bacterium, Erwinia carotovora (Buchon, 2009a). Remarkably, the fly midgut can recover not only from damage, but also from severe induced hyperplasia, such as that caused by ectopic cytokine (Upd) production. Thus, this system is robustly homeostatic (Jiang, 2009).
Each of the three stress conditions that were studied induced all three Upd
cytokines, and genetic tests showed that Upd/Jak/Stat signaling was
both required and sufficient for compensatory ISC division and gut
renewal. Although JNK signaling was also activated in each instance, it was not required for the stem cell response to either EC apoptosis or infection, implying that other mechanisms can sense EC loss and trigger
the cytokine and proliferative responses. JNK signaling may be
important in specific contexts that were not tested, such as following
oxidative stress, which occurs during some infections, activates JNK, and stimulates midgut DNA replication (Biteau, 2008; Jiang, 2009).
Following P. entomophila infection, virtually the entire midgut epithelium could be renewed in just 2-3 days, whereas comparable renewal took more than 3 weeks in healthy flies. Despite this radical acceleration of cell turnover, the relative proportions of the different gut cell types generated (ISC, EB, EE, and EC remained similar to those in midguts undergoing slow, basal turnover. These data suggested that de-differentiation did not occur, and little evidence was obtained of symmetric stem divisions (stem cell duplication) induced by enteric infection. Hence, it is suggested that asymmetric stem cell divisions as described for healthy animals,
together with normal Delta/Notch-mediated differentiation, remain the
rule during infection-induced regeneration. The results obtained
using Reaper to ablate ECs are also consistent with this conclusion, as
are those from detergent-induced midgut regeneration (Jiang, 2009).
Unlike infection, direct genetic activation of JNK or Jak/Stat signaling
promoted large increases not only in midgut mitoses, but also in the
pool of cells expressing the stem cell marker Delta.
Cell type marker analysis discounted de-differentiation of EEs or ECs
as the source of the new stem cells, but the reactivation of EBs as
stem cells seems possible. For technical reasons, no tests were performed
to whether stem cell duplications occur in response to Jak/Stat or JNK
signaling, and this also remains possible. The ability of hyperplastic
midguts to recover to normal following the silencing of cytokine
expression suggests that excess stem cells are just as readily eliminated as they are generated. Further studies are required to understand how midgut
stem cell pools can be expanded and contracted according to need (Jiang, 2009).
How the Upds are induced in the midgut by JNK, apoptosis, or infection remains an open question. Paradoxically, ISC divisions triggered by Reaper required EC apoptosis but not JNK activity, whereas ISC divisions
triggered by JNK did not require apoptosis, and ISC divisions triggered by infection required neither apoptosis nor JNK activity. These incongruent results suggest that different varieties of
gut epithelial stress may induce Upd cytokine expression via distinct
mechanisms. In the case of EC ablation, physical loss of cells from the
epithelium might drive the cytokine response. In the case of infection, it is expected the critical inputs to be the Toll and/or IMD innate
immunity pathways, which signal via NF-kappaB transcription factors.
Functional tests, however, indicated that the Toll and IMD pathways are
required for neither Upd/Jak/Stat induction nor compensatory ISC
mitoses following enteric infection by gram-negative bacteria. Hence, other unknown inputs likely trigger the Upd cytokine response to infection (Jiang, 2009).
Is the cytokine response to infection relevant to normal midgut homeostasis? This seems likely. Low levels of Upd3 expression and Stat signaling are observed in healthy animals, and midgut homeostasis required the IL-6R-like receptor Dome and Stat92E even without infection. Wild Drosophila subsist on a diet of rotting fruit, which is a good source of protein because it is teeming with bacteria and fungi. Given such a diet it seems likely that midgut cytokine signaling is constantly modulated by
ever-present factors that impose dietary stress -- food composition and
commensal microbiota -- even in healthy animals (Jiang, 2009).
Although studies in mammals have yet to unravel the details of a feedback
mechanism underlying gut homeostasis, experimental evidence implies
that such a mechanism exists and involves Cytokine/Jak/Stat signaling.
As in Drosophila, damage to the mouse intestinal epithelium
caused by detergents or infection can stimulate cell proliferation in
the crypts, where stem and transient amplifying cells reside. In a mouse model of detergent (DSS)-induced colitis,
colon epithelial damage caused by DSS allows exposure to commensal
microbes, activating NF-kappaB signaling in resident macrophage-like
Dentritic cells. These cells respond by expressing
inflammation-associated cytokines, one of which (IL-6) activates Stat3
and is believed to promote cell proliferation and regeneration.
Consistent with a functional role for Jak/Stat, disruption of the Stat
inhibitor SOCS3 in the mouse gut increased the proliferative response
to DSS and also increased DSS-associated colon tumorigenesis.
Also pertinent is the presence of high levels of phospho-Stat3 in a
majority of colon cancers, where it correlates with adverse outcome,
and the observation that IL-6 can promote the growth of colon cancer
cells, which are thought to derive from ISCs or transient amplifying
cells.
Increased colon cancer incidence is associated with gut inflammatory
syndromes, such as inflammatory bowel disease (IBD) and Crohn's disease,
which are likely to involve enhanced cytokine signaling. Whether
cytokines mediate gut epithelial turnover in healthy people or only
during inflammation is presently unclear, but it nevertheless seems
likely that the mitogenic role of IL-6-like cytokines and Jak/Stat
signaling in the intestine is conserved from insects to humans (Jiang, 2009).
The connection to inflammation suggests that these findings may also be
relevant to the activity of nonsteroidal anti-inflammatory drugs
(NSAIDs), such as aspirin, ibuprofen, and celecoxib, as suppressors of
colorectal carcinogenesis. These drugs target the cyclooxygenase
activity of prostaglandin H synthases (PGHS, COX), which are
rate-limiting for production of prostaglandin E2, a short-range lipid
signal that promotes inflammation, wound healing, cell invasion,
angiogenesis, and proliferation.
Notably, COX-2 has been characterized as an immediate early gene that
can be induced by signals associated with infection and inflammation,
including the proinflammatory cytokines IL-1beta and IL-6, which activate
NF-kappaB and STAT3, respectively.
Whether prostaglandins mediate the effects of Jak/Stat signaling in the
fly midgut remains to be tested, but insects do produce prostaglandins,
and Drosophila has a functional COX homolog, pxt, whose activity can be suppressed by NSAIDs (Jiang, 2009).
The follicle cells of the Drosophila egg chamber provide an excellent model in which to study modulation of the cell cycle. During mid-oogenesis, the follicle cells undergo a variation of the cell cycle, endocycle, in which the cells replicate their DNA, but do not go through mitosis. Previously, it was shown that Notch signaling is required for the mitotic-to-endocycle transition, through downregulating String/Cdc25, and Dacapo/p21 and upregulating Fizzy-related/Cdh1.
In this paper, it is shown that Notch signaling is modulated by Shaggy and temporally induced by the ligand Delta, at the mitotic-to-endocycle transition. In addition, a downstream target of Notch, tramtrack, acts at the mitotic-to-endocycle transition. It is also demonstrated that the JNK pathway is required to promote mitosis prior to the transition, independent of the cell cycle components acted on by the Notch pathway. This work reveals new insights into the regulation of Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).
Notch controls the mitotic-to-endocycle transition in follicle epithelial cells; Notch pathway activity arrests mitotic cell cycle and promotes endocycles by downregulating string/cdc25 and dacapo/p21, and upregulating fzr/Cdh1. This study identified components regulating this transition, Delta, Shaggy, and Tramtrack. Shaggy and Delta are required for the activation of Notch protein. However, Delta is sufficient to activate Notch in this process, since premature expression of Delta in the germline stops mitotic division of the follicle cells. This study identified Tramtrack as a connection between Notch and the cell cycle regulators stg, fzr, and dap. Loss of Tramtrack function phenocopies the Notch and Su(H) phenotypes; overproliferation and misregulation of cell cycle components. However, high FAS3 expression, indicative of differentiation defects in Notch clones, is not observed in ttk clones, suggesting that Tramtrack might regulate a branch of the Notch pathway specific for cell cycle control. It was also shown that the JNK-pathway is a critical mitosis promoting pathway in follicle cells. Loss of JNK(bsk) or JNKK(hep) activities stop follicle cell mitotic cycles, while loss of JNK promotes premature endocycles. In addition, loss of the negative regulator of the pathway, the phosphatase Puckered, results in a lack of endocycles. However, the Notch-responsive cell cycle targets that, in combination, can induce the mitotic-to-endocycle transition, stg, fzr, and dap, are not regulated by the JNK-pathway (Jordan, 2006).
Notch signaling is highly regulated throughout development. The Notch receptor can be regulated by glycosylation of the extracellular domain, as well as by endocytosis and degradation of the intracellular domain, thus affecting the activity of the pathway. Shaggy has been shown to phosphorylate and thus affect the stability of Notch protein. Normal processing and clearing of Notch protein from the apical surface of follicle cells upon Notch activation does not occur in shaggy clones, indicating that Notch is not normally activated and therefore regulation of the downstream targets does not take place (Jordan, 2006).
In many organisms and tissues the Notch ligands are ubiquitously expressed and thus not likely to regulate Notch pathway activation. However, at the mitotic to endocycle transition, Delta is upregulated in the germline, making ligand expression a likely candidate for regulation of Notch activity. Premature expression of Delta in the germline can cause mitotic division to stop at least one stage earlier than in control ovarioles. Nonetheless, this effect is seen in only half of the ovarioles. Therefore, it is possible that yet another process is regulating Notch activity at the transition in addition to Delta expression. Further testing will determine if endocytosis of Notch might also regulate Notch activity at the mitotic-to-endocycle transition. One possible protein is Numb, which regulates Notch in human mammary carcinomas, indicating that Numb may have a more general role in cell cycle control than just the division of the sensory organ precursors (Jordan, 2006).
The fact that Notch overrides the mitotic activity of the JNK pathway by acting on cell cycle regulators that can induce the mitotic-to-endocycle transition puts further demand on understanding the connection between Su(H) and cell cycle regulators. One such component, the transcription factor Tramtrack, has been identified. Two Tramtrack proteins exist, Ttk69 and Ttk88, both of which are affected by the allele used in these studies. However, staining with antibodies specific to the two forms reveals that only Ttk69 is detectable in the follicle cells and downregulated in Notch clones (Jordan, 2006).
Ttk69 can control proliferation in glial cells, strengthening its candidacy for a critical component between Notch and cell cycle controllers in follicle epithelial cells. In addition, the Ttk-like BTB/POZ-domain zinc-finger transcription repressor in humans is Bcl-6, a protein associated with B-cell lymphomas (Jordan, 2006).
Ttk function in the follicle cell mitotic-to-endocycle transition was analyzed and it has been shown that the Notch-responsive cell cycle components stg, dap, and fzr are responsive to Ttk function. Interestingly, Ttk69 controls the string promoter in the Drosophila eye discs. In the future, it will be important to determine whether Ttk DNA binding sites are found in the Notch-responsive stg promoter as well. In addition, the binding sites of transcription factors that can interact with Ttk will be of interest, since Ttk can act as a DNA binding or non-binding repressor (Jordan, 2006).
Previous work revealed that the JNK pathway is closely connected to cell cycle control. For example, in fibroblasts the JNK pathway is critical for cdc2 expression and G2/M cell cycle progression. In the case of the follicle cell mitotic-to-endocycle transition, it was shown that the JNK pathway is a critical positive controller of the mitotic cycles. Lack of JNK activity leads to a block in mitosis and initiation of premature endocycles. Conversely, lack of the negative regulator of the JNK-pathway, the phosphatase Puckered, results in a loss of endocycles. However, puc mutant clones do not consistently support extra divisions but might induce apoptosis as shown recently in disc clones (Jordan, 2006).
These data are interesting in light of the results showing that the JNK pathway does not control the same cell cycle targets as the Notch pathway, and could be explained by the following hypothesis: the JNK-pathway positively regulates the mitotic cycles prior to stage 6 in follicle epithelial cells. This positive action on mitotic cycles is negatively short-circuited by the direct control of cell cycle regulators by the Notch pathway at stage 6 in oogenesis, resulting in the mitotic-to-endocycle transition. Premature termination of the JNK pathway is sufficient to induce mitotic-to-endocycle transition. However, prolonged JNK activity, while disrupting endocycles, cannot maintain mitotic cycling efficiently, due to Notch action on string, dacapo, and fzr (Jordan, 2006).
What then terminates JNK-pathway activity at stage 6 in oogenesis? Prolonged JNK activity (puc mutant clones) affects endocycles and the expression of pJNK and Puc subsides at stages 6-7; results that both suggest the downregulation of JNK activity at the mitotic-to-endocycle transition. One possibility is that Notch activity downregulates the JNK pathway. However, at least Su(H)-dependent Notch activity does not regulate the JNK pathway, since no effect on puckered expression was observed in Su(H) mutant clones. It is plausible that Su(H)-independent Notch activity regulates the JNK pathway in this context, as has been shown to be the case in dorsal closure. Interestingly, Deltex might play a role in this Su(H)-independent Notch activity (Jordan, 2006).
An important question in analyzing the developmental control of cell cycle is whether the same signaling pathways control both differentiation and cell cycle, and if so, how the labor is divided. The Notch-dependent mitotic-to-endocycle transition is an example of such a question; Notch action in stage 6 follicle cells is critical for the cell cycle switch and for at least some aspects of differentiation. This work reports the first component that separates Notch dependent cell cycle regulation from Fas3 marked differentiation; Ttk. In the ttk mutant clones, upregulation of FAS3, characteristic for Notch clones, is not observed. Therefore, Ttk constitutes a branch of Notch activity that might be solely required for cell cycle control in this context. However, Ttk's independent function cannot yet be rule out. In the future, it will be important to understand whether signaling pathways in general show a clear separation of differentiation and cell cycle control on the level of downstream transcription factors. Importantly, these and previous results have revealed the essential cell cycle regulators and their roles in controlling the Notch-dependent mitotic-to-endocycle switch (Jordan, 2006).
Although bilateral animals appear to have left–right (LR) symmetry from the outside, their internal organs often show directional and stereotypical LR asymmetry. The mechanisms by which the LR axis is established in vertebrates have been extensively studied. However, how each organ develops its LR asymmetric morphology with respect to the LR axis is still unclear. This study showed that Drosophila Jun N-terminal kinase (JNK) signaling is involved in the LR asymmetric looping of the anterior-midgut (AMG) in Drosophila. Mutant embryos of puckered (puc), which encodes a JNK phosphatase, show random laterality of the AMG. Directional LR looping of the AMG requires JNK signaling to be down-regulated by puc in the trunk visceral mesoderm. Not only the down-regulation, but also the activation of JNK signaling is required for the LR asymmetric looping. It was also found that the LR asymmetric cell rearrangement in the circular visceral muscle (CVM) is regulated by JNK signaling and required for the LR asymmetric looping of the AMG. Rac1, a Rho family small GTPase, augments JNK signaling in this process. These results also suggest that a basic mechanism for eliciting LR asymmetric gut looping may be conserved between vertebrates and invertebrates (Taniguchi, 2007).
This report, demonstrates that JNK signaling activity must be controlled properly in CVM cells for normal LR asymmetric development of the AMG. This idea is supported by the following observations. First, in the puc mutant, which has a hyperactivated JNK signal, the LR asymmetry of this organ is random, and the LR defects are rescued by puc expression in the trunk visceral mesoderm. Second, these LR defects are suppressed by the down-regulation of bsk, a Drosophila JNK homolog. Third, a stage-specific suppression of JNK signaling by the overexpression of puc results in laterality defects in the AMG. Importantly, however, there was no LR asymmetry in the expression pattern of the JNK signal or puc before or during the LR asymmetric development of this organ. It is therefore speculated that JNK signaling has a permissive role for LR asymmetric development of the AMG, rather than being an instructive cue for this process. For example, bilateral JNK signaling could have some influence on an instructive LR signaling molecule that has a LR differences in its activity or distribution. In this case, hyperactivation of JNK signaling may bring this instructive LR signal up or down across a threshold for the LR difference, triggering the LR asymmetric development of this organ (Taniguchi, 2007).
In Drosophila, several downstream targets of JNK signaling have been identified and studied extensively, and decapentaplegic and Delta are known downstream target genes of JNK signaling. However, the curret studies, including gene expression and genetic interaction analyses, demonstrate that these genes are unlikely to be involved in the JNK signal-driven LR asymmetric development of the AMG. In addition, previous report showed that the overexpression of Myo31DF inversed the laterality of the AMG. However, it was also found that puc did not influence the laterality defects induced by expressing Myo31DF, suggesting distinct roles for Myo31DF and JNK signaling in this process. In contrast to these observations, the expression of Fas3, which encodes an adhesion molecule localized to septate junctions, is significantly decreased in the CVM of the puc mutant. However, Fas3 is not required for the normal LR asymmetric development of the AMG. Therefore, it is presently unknown which target gene of JNK signaling is involved in the LR asymmetric development of the AMG (Taniguchi, 2007).
In contrast, this study demonstrates that the up-regulation of Rac1 activity in CVM cells results in LR defects of the AMG. In addition, Rac1 functions upstream of JNK in the CVM during LR asymmetric development of the AMG. Thus, the cytoskeletal rearrangement that is modulated by Rac1 upstream of JNK could be important in the LR asymmetric rearrangement of CVM cells. It is known that JNK activates head involution defective (hid) to induce apoptosis in and the clockwise rotation of the terminalia, which arises from the genital disc. In contrast to this organ's requirement for apoptosis for normal LR asymmetric development, this study found that the overexpression of p35, a potent suppressor of hid-dependent apoptosis, in CVM cells does not affect the LR asymmetric development of the AMG. In addition, the increased apoptosis of CVM cells was not observed in pucGS16811 mutant embryos. These results suggest that, unlike its role in the genital disc, apoptosis is not involved in the LR asymmetric rearrangement of CVM cells, although JNK signaling plays essential roles in the LR asymmetric development of both organs (Taniguchi, 2007).
puc was found to be required for normal LR asymmetric development of the AMG at some time during stages 11 to 14. In addition, the LR asymmetric rearrangement of CVM cells is required for the normal laterality of the AMG. However, the LR asymmetric rearrangement of these cells occurs after stage 16. These results suggest that the down-regulation of JNK signaling by puc is required before, rather than during, the LR asymmetric cell rearrangement in the CVM. A possible explanation is that the down-regulation of JNK signaling is required to change the states of these cells to allow them to respond to the LR signal. It is speculated that a small LR bias at the initial stage, as found in CVM cells at late stage 15, is probably sufficient to introduce stereotypic LR asymmetry into the subsequent events of AMG morphogenesis. According to this model, the subsequent rotation of the AMG, which mostly occurs in the dorsoventral direction, augments the initial LR bias. Therefore, if the small bias is not introduced initially, the AMG shows nondirectional LR asymmetry, as was found in the puc mutant embryos (Taniguchi, 2007).
The Drosophila gut consists of two layers of cells, the epidermis and the visceral musculature. It was previously demonstrated that Myo31DF, which is essential for normal laterality of the hindgut, is required in the epithelium of this organ (Hozumi, 2006). This finding suggests that rearrangement of the epithelial cells is responsible for the LR asymmetric development of the embryonic hindgut. In contrast, for the normal LR asymmetric development of the AMG, the surrounding CVM cells play a crucial role (Taniguchi, 2007).
In zebrafish, the looping of the LR asymmetric gut is elicited as a secondary consequence of the LR asymmetric migration of the lateral plate mesoderm. The embryonic gut of this organism is located along the midline between the lateral plate mesoderm of each lateral half. The shape of the space between the left and right lateral plate mesodermal tissue becomes stereotypically LR asymmetrical, as a consequence of LR asymmetric changes in the configuration of these mesodermal tissues. Because the gut lies in the vacant space delineated by these tissues, the morphology of this organ also develops directional LR asymmetry. This study has demonstrated that the mesodermal tissue also plays an essential role in the LR asymmetric development of the Drosophila AMG. Although it is not known whether JNK signaling is involved in the LR asymmetric development of the lateral plate mesoderm in zebrafish, the roles of the mesodermal cells in the LR asymmetric development of the gut may be evolutionarily conserved between vertebrates and insects, at least to some extent (Taniguchi, 2007).
Collective cell movement is a mechanism for invasion identified in many developmental events. Examples include the movement of lateral-line neurons in Zebrafish, cells in the inner blastocyst, and metastasis of epithelial tumors. One key model to study collective migration is the movement of border cell clusters in Drosophila. Drosophila egg chambers contain 15 nurse cells and a single oocyte surrounded by somatic follicle cells. At their anterior end, polar cells recruit several neighboring follicle cells to form the border cell cluster. By stage 9, and over 6 hr, border cells migrate as a cohort between nurse cells toward the oocyte. The specification and directionality of border cell movement are regulated by hormonal and signaling mechanisms. However, how border cells are held together while they migrate is not known. This study shows that negative-feedback loop controlling JNK activity regulates border cell cluster integrity. JNK signaling modulates contacts between border cells and between border cells and substratum to sustain collective migration by regulating several effectors including the polarity factor Bazooka and the cytoskeletal adaptor D-Paxillin. A role for the JNK pathway is anticipated in controlling collective cell movements in other morphogenetic and clinical models (Llense, 2008).
In an analysis of the mechanisms regulating the expression of puckered (puc), the gene encoding the Drosophila Jun N-terminal kinase (JNK) dual-specificity phosphatase (DSP) (Martin-Blanco, 1998) regulatory sequences (PG2) were uncovered directing its expression to border cells. PG2 expands across the first and second introns of puc, in which the pucB48 insertion is located. This expression is also observed in puc enhancer (pucB48) and protein trap lines (Llense, 2008).
JNKs represent a signaling hub with pivotal functions in cell proliferation, differentiation, and death. JNKs are inactivated by DSPs, and transcriptional induction of DSP expression is well documented as a negative-feedback mechanism. In Drosophila, this loop modulates JNK activity in processes such as epithelial expansion and overexpression of dominant-negative constructs relies on JNK signaling. Further, Puc overexpression leads to inhibition of JNK activity. Thus, Puc implements a negative-feedback loop in border cells (Llense, 2008).
Defects caused by the loss of JNK function in border cells included cluster dissociation and impaired motility. Instead of collectively following a leader cell, JNK-minus border cells autonomously disperse at the late step of migration, with most exhibiting long cellular extensions (LCEs) and actin-rich protrusions. JNK signaling does not affect polar cell specification or border cell recruitment (Llense, 2008).
Dissociation phenotypes are also observed in JNK-specific but not ERK-specific loss-of-function conditions for D-Fos, a major MAPK target, thereby ruling out potential interference via ERK. Indeed, reduced D-Fos suppresses border cell migration defects induced by elevated JNK activity (Llense, 2008).
Does JNK act in a linear pathway or does it target multiple independent effectors simultaneously to produce a multifaceted phenotype? Cells that migrate as part of a group cling firmly to each other while adhering transiently to the substrate. So, during migration, border cells show apicobasal polarity and remain attached to one another and to polar cells. Cell contacts are enriched in the adherens junctions (AJs) components, DE-Cadherin and Armadillo (β-Catenin). In electron microscopy (EM) preparations, border cells are tightly bound, whereas interfaces between border and nurse cells exhibit multiple interdigitations (Llense, 2008).
In JNK-minus conditions, namely after Puc overexpression or in bsk (JNK) clones, cell polarity is disrupted and only remnants of apical markers, such as Bazooka (Baz), are present. Adhesion is impaired, and DE-Cadherin and Armadillo are downregulated. Reduction of JNK activity also resulted in β-Integrin accumulation at ectopic actin-rich protrusions. These also accumulate MyoVI, consistent with its role in force generation. In summary, upon depletion of JNK activity, border cells lose apicobasal polarity and progress into a mesenchymal phenotype. Indeed, EM preparations show that border-border cell contacts are less tight than wild-type cell contacts and cell membranes detach from each other at multiple sites. The end result is a cluster with multiple leading edges and residual cell-cell contacts (Llense, 2008).
How does the JNK pathway become activated in border cells? Rho, Rac, and Cdc42 GTPases are potential candidates. Loss of Rac completely abolishes border cell migration. However, phenotypes for RhoA and Cdc42 expression of dominant-negative forms -- RhoADN and Cdc42DN) closely resemble JNK-minus induced dissociation. Furthermore, in Cdc42DN, polarity, cell contacts, and redistribution of substrate adhesion and motor markers are similarly affected. Most importantly, reporters of JNK activity such as Jun phosphorylation and the expression of the pucB48 transgene are also downregulated. Null cdc42 MARCM clones display the same phenotype, although frequency and penetrancy were very low. Therefore, a role for other GTPases, such as RhoA, in JNK activation cannot be ruled out (Llense, 2008).
Border cell clusters deficient for Baz (BazRNAi) resemble JNK loss of function (which leads to Baz downregulation) and exhibit dissociation and downregulation of DE-Cadherin. Thus, Baz, a critical landmark of epithelial polarity, could serve as an effector for the control of border-border cell contacts. To test this, Baz was overexpressed in cells lacking JNK activity (or expressing Cdc42DN); Baz was strongly rescued cluster integrity and DE-Cadherin expression (Llense, 2008).
Epithelial cells use a specialized repertoire of integrin receptors to mediate contacts and migration. However, border cells lacking β-Integrin were still able to adopt a leading migratory position, although the effect of complete removal of integrins from the cluster has not been reported (Llense, 2008).
Interestingly, β-Integrin antibodies reveal a rosette staining in border cell clusters that colocalize with AJ markers. Thus, β-Integrin could participate in the stabilization or strengthening of cell contacts, as shown for amnioserosa and larval epithelial cells in Drosophila, mammalian keratinocytes, and carcinoma cell clusters. Furthermore, β-Integrin, after JNK inactivation, strikingly accumulates at the front of LCEs suggesting a second function in cell invasiveness, as observed in leukocytes (Llense, 2008).
Direct evidence for β-Integrin involvement in border cell migration was obtained by RNAi in a sensitized JNK-minus condition. The expression of β-Integrin dsRNAs in border cells reduced β-Integrin levels but did not cause migration or integrity defects. However, in the presence of Puc, β-Integrin RNAi led to a strong enhancement of cluster dissociation and prevented the full extension of LCEs, which become mostly blunted. Moreover, an adhesion dominant negative (diβ) integrin chimera showed weak, but reproducible, dissociation phenotypes. Thus, β-Integrin turns out to participate in, first, the stabilization of border-border cell contacts and, second, the promotion of LCEs extension. The integrin countereceptors that facilitate border cell attachment and invasiveness are not yet known (Llense, 2008).
D-Paxillin was present in border cell contacts but was downregulated in JNK-minus conditions. Genomic-profiling analyses of JNK mutants suggests a transcriptional control of D-Paxillin expression. However, other options, such as subcellular relocation after phosphorylation, could also explain why D-Paxillin may be absent from JNK-minus border cells. Expression in border cells of two different D-Paxillin dsRNA lines was found to result in JNK loss-of-function-like dissociation, DE-Cadherin downregulation and β-Integrin accumulation at LCEs. Expression of a Talin RNAi line does not produce any migration phenotype, although it impairs follicle epithelia integrity (Llense, 2008).
In migratory leukocytes, PKA-mediated integrin phosphorylation prevents Paxillin accumulation at the leading front. Paxillin-integrin interactions in lateral positions lead to the inhibition of Rac, whose activation is thus spatially limited to the leading edge where it induces lamellipodia. Consequently, D-Paxillin might stabilize β-Integrin in border-border cell contacts. Its absence, in JNK-minus conditions, would lead in lateral and trailing cells to Rac activation, dissociation of border-border cell contacts, and extension of β-Integrin-rich ectopic lamellipodia. Indeed, the PKA-RII subunit is expressed in border cells, and border cells mutant for PKA show migration defects (Llense, 2008).
Interestingly, D-Paxillin overexpression rescued the border cell defect resulting from loss of JNK activity (or expression of Cdc42DN). DE-Cadherin relocated to border-border cell contacts, and β-Integrin expression was partially eliminated from residual LCEs. D-Paxillin overexpression alone had no effects (Llense, 2008).
It was further asked whether the control of cell polarity and cytoskeletal adaptor proteins by JNK were related. Paxillin expression was strongly reduced in baz mutant conditions, whereas Baz expression was only slightly affected by interference in Paxillin expression (Llense, 2008).
JNK signaling regulates border cells clustering by controlling at least two key elements, cell polarity (Baz) and cytoskeletal adaptor proteins (D-Paxillin), and as a consequence cell-cell contacts and cell-substrate attachments. Interestingly, the overexpression of Hindsight (Hnt), a target and negative regulator of JNK, results in similar defects to those caused by inhibition of JNK. Because re-expression of a variety of proteins (Baz, D-Paxillin, DE-Cadherin, and Armadillo) can rescue the dissociation phenotype and given that each time rescue is achieved, DE-Cadherin and Armadillo expression are restored, a plausible explanation for the effects observed with JNK-minus and Hnt overexpression is that there is an overall loss of multiple cell-cell adhesion complexes. The restoration of any of them would provide sufficient cell-cell adhesion to enable the cluster to move as a collective (Llense, 2008).
The individual migratory abilities of JNK-minus border cells could be partially explained by the observed β-Integrin relocalization to LCEs (border-nurse cell contacts). Alternatively, border cells could have lost their capacity to respond to positional gradients leading to random outward movements. Border cells use PVF and EGF to guide their migration. Blocking PVR and EGFR does not reduce the ability of border cells to extend protrusions but abolishes their directionality, with protrusions now extending in all directions. However, in these conditions, border cell clusters do not dissociate, thereby ruling out the possibility that dissociation in JNK mutants is due only to loss of directional guidance. A directionality index (DI) can be calculated. A DI of 0 indicates equal numbers of protrusions extending forward and backward. A DI of 1 indicates that cells only extend protrusions in the direction of migration. This study found a DI of 0.59 for wild-type clusters. In the absence of JNK, however, clusters show a DI ranging from -0.2 to 0, suggesting that JNK-minus border cells are blind to positional cues. This fact accounts for recently described synergistic effects of JNK and PVR signaling on border cells (Llense, 2008).
This model makes a significant prediction: JNK hyperactivation should increase adhesiveness and eventually block migration. Accordingly, ut was observed that the overexpresssion of a constitutively active form of Hep, the overexpresssion of a constitutively active form of Misshapen, or loss of function clones of puc resulted in nonmigratory and strongly compacted clusters. Occasionally, the death of a number of border cells was observed (Llense, 2008).
So far, the molecular and cellular study of collective versus individual migration both in developmental and cancer models has mainly focused on the analysis of structural elements. The identification of the JNK cascade as a key determinant of migratory responses in border cells could have an important impact in the understanding of collective movements. Border cell migration could serve as a good model for studying migratory transitions, thus impacting on the understanding of cancer metastasis and invasiveness, during which so little is known about the signaling mechanisms controlling migratory behavior (Llense, 2008).
Reactive oxygen species (ROS), produced during various electron transfer reactions in vivo, are generally considered to be deleterious to cells. In the mammalian haematopoietic system, haematopoietic stem cells contain low levels of ROS. However, unexpectedly, the common myeloid progenitors (CMPs) produce significantly increased levels of ROS. The functional significance of this difference in ROS level in the two progenitor types remains unresolved. This study shows that Drosophila multipotent haematopoietic progenitors, which are largely akin to the mammalian myeloid progenitors, display increased levels of ROS under in vivo physiological conditions, which are downregulated on differentiation. Scavenging the ROS from these haematopoietic progenitors by using in vivo genetic tools retards their differentiation into mature blood cells. Conversely, increasing the haematopoietic progenitor ROS beyond their basal level triggers precocious differentiation into all three mature blood cell types found in Drosophila, through a signalling pathway that involves JNK and FoxO activation as well as Polycomb downregulation. It is concluded that the developmentally regulated, moderately high ROS level in the progenitor population sensitizes them to differentiation, and establishes a signalling role for ROS in the regulation of haematopoietic cell fate. These results lead to a model that could be extended to reveal a probable signalling role for ROS in the differentiation of CMPs in mammalian haematopoietic development and oxidative stress response (Owusu-Ansah, 2009).
The Drosophila lymph gland is a specialized haematopoietic organ which produces three blood cell types -- plasmatocytes, crystal cells and lamellocytes -- with functions reminiscent of the vertebrate myeloid lineage. During the first and early second larval instars, the lymph gland comprises only the progenitor population. However, by late third instar, multipotent stem-like progenitor cells become restricted to the medial region of the primary lymph gland lobe, in an area referred to as the medullary zone; whereas a peripheral zone, referred to as the cortical zone, contains differentiated blood cells. By late third instar, the progenitors within the medullary zone are essentially quiescent, whereas the mature, differentiated population in the cortical zone proliferates extensively. The posterior signalling centre is a group of about 30 cells that secretes several signalling molecules and serves as a stem-cell niche regulating the balance between cells that maintain 'stemness' and those that differentiate (Owusu-Ansah, 2009).
Although several studies have identified factors that regulate the differentiation and maintenance of Drosophila blood cells and the stem-like progenitor population that generates them, intrinsic factors within the stem-like progenitors are less explored. Interrogation of these intrinsic factors is the central theme of this investigation. It was observed that by the third instar, the progenitor population in the normal wild-type lymph gland medullary zone contains significantly increased ROS levels compared with their neighbouring differentiated progeny that express mature blood cell markers in the cortical zone. ROS are not increased during the earlier larval instars but increase as the progenitor cells become quiescent and subside as they differentiate. This first suggested that the rise in ROS primes the relatively quiescent stem-like progenitor cells for differentiation. ROS was reduced by expressing antioxidant scavenger proteins GTPx-1 or catalase, specifically in the progenitor cell compartment using the GAL4/UAS system, and it was found that suppressing increased ROS levels in haematopoietic progenitors significantly retards their differentiation into plasmatocytes. As a corollary, mutating the gene encoding the antioxidant scavenger protein superoxide dismutase (Sod2) led to a significant increase in differentiated cells and decrease in progenitors (Owusu-Ansah, 2009).
ROS levels in cells can be increased by the genetic disruption of complex I proteins of the mitochondrial electron transport chain, such as ND75 and ND42. Unlike in wild type, where early second-instar lymph glands exclusively comprise undifferentiated cells, mitochondrial complex I depletion triggers premature differentiation of the progenitor population. This defect is even more evident in the third instar, where a complete depletion of the progenitors is seen as primary lobes are populated with differentiated plasmatocytes and crystal cells. The third differentiated cell type, the lamellocyte, defined by the expression of the antigen L1, is rarely observed in the wild-type lymph gland but is abundantly seen in the mutant. Finally, the secondary and tertiary lobes, largely undifferentiated in wild type, also embark on a robust program of differentiation upon complex I depletion. Importantly, the phenotype resulting from ND75 disruption can be suppressed by the co-expression of the ROS scavenger protein GTPx-1, which provides a causal link between increased ROS and the premature differentiation phenotype. It is concluded that the normally increased ROS levels in the stem-like progenitors serve as an intrinsic factor that sensitizes the progenitors to differentiation into all three mature cell types. Any further increase or decrease in the level of ROS away from the wild-type level enhances or suppresses differentiation respectively (Owusu-Ansah, 2009).
In unrelated systems, increased ROS levels have been demonstrated to activate the JNK signal transduction pathway. Consequently, it was tested whether the mechanism by which the progenitors in the medullary zone differentiate when ROS levels increase could involve this pathway. The gene puckered (puc) is a downstream target of JNK signalling and its expression has been used extensively to monitor JNK activity. Although puc transcripts are detectable by reverse transcriptase PCR (RT- PCR), the puc-lacZ reporter is very weakly expressed in wild type. After disruption of ND75, however, a robust transcriptional upregulation of puc-lacZ expression can be seen, indicating that JNK signalling is induced in these cells in response to high ROS levels. The precocious progenitor cell differentiation caused by mitochondrial disruption is suppressed upon expressing a dominant negative version of basket (bsk), the sole Drosophila homologue of JNK. This suppression is associated with a decrease in the level of expression of the stress response gene encoding phosphoenol pyruvate carboxykinase; quantitatively a 68% suppression of the ND75 crystal cell phenotype was observed when JNK function was removed as well. Although disrupting JNK signalling suppressed differentiation, ROS levels remain increased in the mutant cells, as would be expected from JNK functioning downstream of ROS (Owusu-Ansah, 2009).
In several systems and organisms, JNK function can be mediated by activation of FoxO as well as through repression of Polycomb activity. FoxO activation can be monitored by the expression of its downstream target Thor, using Thor-lacZ as a transcriptional read-out. This reporter is undetectable in wild-type lymph glands although Thor transcripts are detectable by
RT-PCR; however, the reporter is robustly induced when complex I is disrupted, suggesting that the increase in ROS that is mediated by loss of complex I activates FoxO. To monitor Polycomb de-repression, a Polycomb reporter was used that expresses lacZ when Polycomb proteins are downregulated. Although undetectable in wild-type lymph glands, disrupting ND75 leads to lacZ expression suggesting that Polycomb activity is downregulated by the altered ROS and resulting JNK activation. Direct FoxO overexpression causes a remarkable advancement in differentiation to a time as early as the second instar, never seen in wild type. By early third instar, the entire primary and secondary lobes stained for plasmatocyte and crystal cell markers when FoxO is expressed in the progenitor population. Unlike with ROS increase, no a significant increase in lamellocytes was found upon FoxO overexpression. However, downregulating the expression of two polycomb proteins, Polyhomeotic Proximal (Php-x) and Enhancer of Polycomb [E(Pc)], that function downstream of JNK, markedly increased lamellocyte number without affecting plasmatocytes and crystal cells. When FoxO and a transgenic RNA interference (RNAi) construct against E(Pc) are expressed together in the progenitor cell population, differentiation to all three cell types is evident. It is concluded that FoxO activation and Polycomb downregulation act combinatorially downstream of JNK to trigger the full differentiation phenotype: an increase in plasmatocytes and crystal cells due to FoxO activation, and an increase in lamellocytes primarily due to Polycomb downregulation (Owusu-Ansah, 2009).
This analysis of ROS in the wild-type lymph gland highlights a previously unappreciated role for ROS as an intrinsic factor that regulates the differentiation of multipotent haematopoietic progenitors in Drosophila. Any further increase in ROS beyond the developmentally regulated levels, owing to oxidative stress, will cause the progenitors to differentiate into one of three myeloid cell types. It has been reported that the ROS levels in mammalian haematopoietic stem cells is low but that in the CMPs is relatively high. The Drosophila haematopoietic progenitors give rise entirely to a myeloid lineage and therefore are functionally more similar to CMPs than they are to haematopoietic stem cells. It is therefore a remarkable example of conservation to find that they too have high ROS levels. The genetic analysis makes it clear that the high ROS in Drosophila haematopoietic progenitors primes them towards differentiation. It will be interesting to determine whether such a mechanism operates in mammalian CMPs. In mice, as in flies, a function of FoxO is to activate antioxidant scavenger proteins. Consequently, deletion of FoxO increases ROS levels in the mouse haematopoietic stem cell and drives myeloid differentiation. However, even in the mouse haematopoietic system, FoxO function is dose and context dependent, as ROS levels in CMPs are independent of FoxO. Thus, although the basic logic of increased ROS in myeloid progenitors is conserved between flies and mice, the exact function of FoxO in this context may have diverged (Owusu-Ansah, 2009).
Past work has hinted that ROS can function as signalling molecules at physiologically moderate levels. This work supports and further extends this notion. Although excessive ROS is damaging to cells, developmentally regulated ROS production can be beneficial. The finding that ROS levels are moderately high in normal Drosophila haematopoietic progenitors and mammalian CMPs raises the possibility that wanton overdose of antioxidant products may in fact inhibit the formation of cells participating in the innate immune response (Owusu-Ansah, 2009).
In metazoan development, the precise mechanisms that regulate the completion of morphogenesis according to a developmental timetable remain elusive. The Drosophila male terminalia is an asymmetric looping organ; the internal genitalia (spermiduct) loops dextrally around the hindgut. Mutants for apoptotic signaling have an orientation defect of their male terminalia, indicating that apoptosis contributes to the looping morphogenesis. However, the physiological roles of apoptosis in the looping morphogenesis of male terminalia have been unclear. This study shows the role of apoptosis in the organogenesis of male terminalia using time-lapse imaging. In normal flies, genitalia rotation accelerates as development proceeded, and completes a full 360° rotation. This acceleration is impaired when the activity of caspases or JNK or PVF/PVR signaling was reduced. Acceleration was induced by two distinct subcompartments of the A8 segment that form a ring shape and surround the male genitalia: the inner ring rotates with the genitalia and the outer ring rotates later, functioning as a 'moving walkway' to accelerate the inner ring rotation. A quantitative analysis combining the use of a FRET-based indicator for caspase activation with single-cell tracking showed that the timing and degree of apoptosis correlates with the movement of the outer ring, and upregulation of the apoptotic signal increases the speed of genital rotation. Therefore, apoptosis coordinates the outer ring movement that drives the acceleration of genitalia rotation, thereby enabling the complete morphogenesis of male genitalia within a limited developmental time frame (Kuranaga, 2011).
To visualize the genitalia rotation in living animals, the His2Av-mRFP Drosophila line was used whose nuclei are ubiquitously marked by a fluorescent protein. The genital disc is a compound disc comprised of cells from three different embryonic segments: A8 (male eighth tergite), A9 (male primordium) and A10 (anal). During metamorphosis, the genital disc is partially everted, exposing its apical surface, and adopts a circular shape. The results captured the male genitalia undergoing a 360° clockwise rotation. Inhibiting apoptosis by expressing the baculovirus pan-caspase inhibitor p35 driven by engrailed-GAL4 (en-GAL4), which is expressed in the posterior compartment of each segment, results in genital mis-orientation at the adult stage (Kuranaga, 2011).
In flies expressing nuclear fluorescent protein driven by en-GAL4, it was observed that the posterior part of the A8 segment (A8p) formed a ring of cells surrounding the A9-A10 part of the disc. First, the images were recorded at a low resolution (10× objective lens) to measure the rotation speed accurately in control and p35-expressing flies, because long-term time-lapse imaging at a high resolution can cause photodamage, and thus alter pupal development. Most of the cells in the A8p that seem to disappear actually moved out of the plane of focus. The imaging results, the rotation started around 24 hours APF (after puparium formation) and stopped about 12 hours later. To confirm whether the mis-oriented genital phenotype in the caspase-inhibited flies was caused by incomplete rotation, the rotation was observed in flies expressing p35 under the en-GAL4 driver. In the p35-expressing flies, the rotation began, but it stopped before it was complete, after about 12 hours, i.e. with the same timing as in control flies. This suggested that the reduced caspase activation in A8p prevented the genitalia from completing the rotation, resulting in mis-oriented adult genitalia (Kuranaga, 2011).
To compare complete rotation with incomplete rotation, the rotation speed was calculated by measuring the angle (thetacontrol and thetap35) of the A9 genitalia every 30 minutes on time-lapse images. The normal rotation was composed of at least four steps: initiation, acceleration, deceleration and stopping. The velocity of rotation V=dtheta/dt was calculated by measuring theta as a function of time t. At first, the genitalia rotated at an average velocity (Vcontrol) of 7.67±3.72°/hour by 1 hour after initiation, then the rotation accelerated, with Vcontrol gradually increasing to 53.83±7.11°/hour by 7 hours after initiation. Interestingly, in the p35-expressing flies, the rotation normally started at 24 hours APF, and the average velocity (Vp35) from the initial rotation to 1 hour later was 7.45± 2.98°/hour, which was not significantly different from the normal rotation. However, the acceleration of the rotation in the p35-expressing flies was lower than normal, with Vp35 gradually increasing to 21.35±7.45°/hour at 5.5 hours after initiation. The first peak of the acceleration rate, which was defined as the initiation of rotation, was observed in the p35-expressing flies (ap35) and was the same as in the control flies (acontrol). However, the duration of the acceleration period was shorter in the p35-expressing flies. These data suggest a relationship between apoptosis and the acceleration of genitalia rotation (Kuranaga, 2011).
Next, the signaling mechanism(s) involved in the acceleration of genitalia rotation wee examined. The inhibition of JNK (c-Jun N-terminal kinase) and PVF (platelet vascular factor) signaling in male flies has been shown to result in mis-oriented adult male terminalia, and it has been hypothesized that the PVF/PVR (PVF receptor) may affect the genitalia rotation via JNK-mediated apoptosis (see Benitez, 2010). Consistent with previous reports, the acceleration of genitalia rotation was significantly impaired in flies expressing dominant-negative forms of JNK (JNK-DN) and PVR (PVR-DN). These data implied that caspase activation and JNK signaling contribute to driving the acceleration of genitalia rotation (Kuranaga, 2011).
To analyze how the genitalia accelerate their rotation, the movement of A8p was traced at the single-cell level. For this experiment, live imaging was performed at a high resolution (20× objective lens), which enabled the cells in A8p to be tracked at single-cell resolution. Cells that were neighbors of A9 rotated with A9, whereas cells located in the anterior half of A8p rotated later than A9. Based on this imaging, A8p was divided into two sheets, named A8pa (anterior of A8p) and A8pp (posterior of A8p). It was found that a part of the cells in A8p underwent apoptosis (Kuranaga, 2011).
To observe caspase activation in living animals, a FRET (fluorescence resonance energy transfer)-based indicator, SCAT3 (sensor for activated caspases based on FRET) was generated. To precisely evaluate apoptosis, a nuclear localization signal-tagged SCAT3 (nls-SCAT3; UAS-nls-ECFP-venus) was used. The nls-SCAT3 signal was clearly observed in A8p. Cells exhibiting high caspase activity were extruded into the body cavity and disappeared, consistent with their apoptotic death and engulfment by circulating hemocytes. Each cell was tracked in the A8p region during the first half of the rotation, and it was found that at least three types of cellular behavior were observed: (1) cells located in A8pp moved with A9, (2) cells underwent apoptosis and (3) cells located in A8pa rotated later (Kuranaga, 2011).
Thus, to observe the behavior of the cells in A8pa, Abdominal B (AbdB) was used as an A8 marker. AbdB is a homeotic gene that is required for the correct development of the genital disc, and AbdB-GAL4LDN is expressed in the segment A8 (in A8a and A8p) of the genital disc during the 3rd instar larval stage. At 24 hours APF, AbdB was expressed in A8 and formed a ring. Time-lapse images were taken, and unexpectedly it was found that most of the cells in the AbdB-expressing region underwent a 180° clockwise movement, suggesting that AbdB was not expressed in the A8pp region that moved 360° with A9. To determine the speed of the AbdB-expressing cells, three individual cells were traced in each fly, and the value of the turning angle of the cells (thetaAbdB) was calculated. The findings confirmed that the AbdB-expressing region moved halfway around. Although cells in the AbdB-expressing region moved only 180°, the A8pp (inner ring), which was encircled by the AbdB-expressing region (outer ring), still moved 360°. Furthermore, the imaging data indicated that the movement of the outer ring started 1-2 hours later than that of the A9 region, when the acceleration of the genitalia rotation occurred. These observations raise the possibility that the outer ring movement is related to the acceleration of the genitalia rotation (Kuranaga, 2011).
It was therefore considered that the outer ring movement was restricted in the p35-expressing flies, resulting in an incomplete genitalia rotation of about 180°, which mimics the movement of only the inner ring. To verify this possibility, the movement of the outer ring was examined in the p35-expressing flies (en-GAL4+UAS-p35). Although the inner ring rotated normally, the rotation of the outer ring was impaired in the p35-expressing flies. The turning angles were determined by tracing cells in the p35-expressing flies and it was found that thetap35 _inner increased, while the increase of thetap35 _outer was impaired. These data suggest that the A8 segment is composed of two independently regulated rings, and when apoptosis is inhibited, the inner ring can move only 180° with no outer ring movement, resulting in incomplete genitalia rotation (Kuranaga, 2011).
Thus, to determine whether apoptosis correlates with the outer ring movement, the apoptosis was quantified in A8pa every 10 minutes from 0-8 hours after the start of genitalia rotation. The frequency of apoptosis (Rapoptosis) was normalized to the total number of apoptotic cells in each individual. Pulsatile increases in Rapoptosis were observed, with peaks at 1, 2.5 and 4 hours after the start of genitalia rotation. To verify the participation of Rapoptosis in the initiation of outer ring movement, the acceleration rate of thetaAbdB (aAbdB) was calculated by measuring VAbdB as a function of time t, and Rapoptosis was compared with aAbdB. The starting time of outer ring movement was characterized by the early peaks of aAbdB. The analysis suggested that the aAbdB was related to the Rapoptosis, because aAbdB showed its first two peaks at about 1 and 2.5 hours after genitalia rotation started. To quantify these observations, the correlation was calculated between Rapoptosis and aAbdB. This analysis confirmed that there was a strong correlation between these parameters, because the correlation between aAbdB and Rapoptosis is approximately linear during this time. Therefore, these data implied a possible mechanism of apoptosis that facilitates the outer ring movement (Kuranaga, 2011).
To verify this possibility, whether the upregulation of apoptotic signals induces an increase in genitalia rotation speed was meastured. Because the expression of apoptotic genes using an en-GAL4 driver, which is expressed at the embryonic stage, is lethal, the TARGET system was used to control gene expression temporally. Flies were allowed to develop at 18°C until the head of the pupae had just everted, to inhibit gene expression. The pupae were then heat-shocked at 29°C for 12 hours to induce gene expression. Live imaging was performed at 22°C, after the heat shock. At this temperature, the genitalia rotation in the control flies was slower than in control flies bred at 25°C, because a low breeding temperature affects the rate of fly development, including genitalia rotation. Therefore, it was necessary in this experiment to compare the rotation speeds at the same temperature. The expression of reaper (rpr), a pro-apoptotic gene, using the TARGET system, showed that the upregulation of apoptotic signaling significantly increased the timing of acceleration and speed of genitalia rotation. These observations led to the proposal that the outer ring functions like a 'moving walkway' to accelerate the speed of the inner part of the structure, including the A9 genitalia, enabling genitalia to complete rotation within the appropriate developmental time window (Kuranaga, 2011).
According to these observations, it was found that apoptosis drives the movement of cell sheets during the morphogenesis of male terminalia. Further questions remain with regard to how apoptosis contributes to the cell sheet movement. A recent study indicated the possibility that local apoptosis acts as a brake release to regulate genitalia rotation, coupled with left-right determination (Suzanne, 2010). However, it has been reported that the cell shape change by apoptosis enables not only the extrusion of dying cells, but also the reorganization of the actin cytoskeleton in neighboring cells. Therefore, apoptosis could affect the behavior of neighboring cells, to act as a main driving force of the cell-sheet movement. Taken together, apoptosis may generally participate in the morphogenetic process of cell-sheet movement during morphogenesis (Kuranaga, 2011).
Drosophila is a leading genetic model system in nervous system development and disease research. Using the power of fly genetics in traumatic axonal injury research will significantly speed up the characterization of molecular processes that control axonal regeneration in the CNS. A versatile and physiologically robust preparation has been developed for the long-term culture of the whole Drosophila brain. This method was used to develop a novel Drosophila model for CNS axonal injury and regeneration. Similar to mammalian CNS axons, injured adult wild-type fly CNS axons fail to regenerate, whereas adult-specific enhancement of protein kinase A activity increases the regenerative capacity of lesioned neurons. Combined, these observations suggest conservation of neuronal regeneration mechanisms after injury. This model was developed to explore pathways that induce robust regeneration; adult-specific activation of c-Jun N-terminal protein kinase signaling was found to be sufficient for de novo CNS axonal regeneration injury, including the growth of new axons past the lesion site and into the normal target area (Ayaz, 2008).
The first models for axonal severing in the CNS of the fly have been developed and have unveiled a marked conservation in the molecular mechanisms underlying neuronal responses to injury in flies and mammals (Leyssen, 2007). Although these paradigms hold promise to significantly enhance insights into axonal degeneration, they do not allow the evaluation of axonal regeneration. Increased insight into the process of regeneration is crucial from a therapeutic point of view that aims at stimulating axons to reconnect to their postsynaptic target. This work describes a protocol that allows, in addition to a versatile series of manipulations, the precise and reliable severing of sLNv axons in explanted adult Drosophila brains. This technique provides the opportunity to study axonal regeneration in the Drosophila CNS and use genetic screens to identify factors that promote axonal regeneration (Ayaz, 2008).
The regenerative responses of injured axons in Drosophila brain explants show remarkable similarities to those seen in mammals. First, the distal, severed ends of the axons became fragmented in a manner that is reminiscent of Wallerian degeneration in mammals, as has been described also in living Drosophila. In both cases, unconnected axons rapidly split into small vesicles, which then disappear. A major advantage of the explant model is that it also allows the study of the proximal, injured axon. One day after injury, the proximal axonal stump forms a bulbar structure that shows morphological similarities to the dystrophic retraction bulbs seen in injured mammalian CNS axons. This structure is extensively remodeled and develops small, and occasionally longer, thin axonal sprouts. Similar morphological changes have been described recently in severed, GFP-marked axons of the mouse spinal cord (Ayaz, 2008).
The lack of efficient regeneration of injured Drosophila axons in brain explants is not attributable to the experimental setup. (1) sLNv neurons in the preparation appear healthy both morphologically and functionally. (2) Axons in explanted larval brains do form elaborate growth cones and grow large distances over the course of a few days. (3) The proximal axonal stumps are clearly dynamic and capable of sending out axonal sprouts. It is therefore suggested that the lack of efficient sLNv axonal regeneration is an intrinsic feature of the adult fly brain. Indeed, the adult-specific, cell-autonomous manipulation of the catalytic activity of PKA can stimulate a generally dormant regrowth capacity. A significantly larger proportion of lesioned brains become capable of extending new axons at the cut tip. This is very similar to the mammalian cAMP-based enhancement of regrowth. In both cases, regrowth is achieved by increasing PKA catalytic activity, either reducing the negative regulation of PKAc through adding of cAMP in the mammalian studies or overexpressing a catalytic mutant version of PKA that is free from endogenous inhibition. In summary, the data presented in this study support the concept that the fly CNS is closer in its response to injury to the mammalian CNS than the mammalian PNS. The mammalian PNS regenerates spontaneously, whereas, in contrast, both the fly and mammalian CNS fail to regenerate except when additional signals are activated. In the case of PKA, the same signal enhances regeneration in both models. This, in addition to several other details, such as the anatomy of the retracting injured axons and the appearance of a lesion gap, support the conservation of key injury response mechanisms between the two CNS systems (Ayaz, 2008).
In mammalian models for CNS regeneration, several modes of neuronal repair are proposed. These neuronal recoveries can be accomplished directly by either the transected axons itself or by sprouts from unlesioned axons in the neighborhood into the denervated target area. However, what is defined as true axonal regeneration is the reentry of the cut axons themselves onto the denervated target area. Axonal regeneration encompasses several types of neuronal responses that can be grossly divided into the following steps: (1) regrowth of the cut axons, (2) guidance of the newly growing axons toward the normal area of innervation, and finally (3) reinnervation of the denervated target area (Ayaz, 2008).
Despite the promising results of PKA manipulation, a constitutively active form of PKA is not sufficient to produce axons that can extend into the original target area. Therefore the hypothesis was tested that JNK signaling might support better regeneration. The data suggest that JNK is likely to be a key pathway in CNS axonal regeneration after injury. Inhibiting JNK activity results in a high tendency to inhibit the length of the newly grown axons compared with control brains, whereas its constitutive activation results in a massive increase in the penetrance and extent of regeneration, with one-third of all brains showing regenerated axons reentering the target area. It is surprising that activation of only a single pathway is sufficient to enhance the three major anatomical criteria of regeneration. This may be explained by at least two alternative models. It may be that LNv neurons, and presumably other CNS neurons as well, retain their proper guidance and targeting signals well into adult life, and as such genetic manipulations stimulating the frequent growth of lengthy axons can result in axons reaching the correct target area. Alternatively, JNK activation may act as a reprogramming signal allowing neurons to reactivate their specific developmental pathways. Although a role for JNK in axonal navigation cannot be excluded, previous work shows that JNK can stimulate different cell types to grow longer axons, without influencing their guidance properties. In this context, it may seem surprising that the induction of JNK signaling observed during axonal injury is not sufficient to induce regeneration. However, it is in fact likely that the transient upregulation of JNK does play a role in stimulating growth after injury because, when injured axons express dominant-negative forms of JNK, they show a strongly reduced regrowth tendency compared with wild-type brains, although this reduction remained just below statistical significance. The reason why activated JNK signaling has such a dramatic influence on regeneration is twofold. First, an activated form of the protein is no longer subject to endogenous negative regulation. Second, this form is expressed as a UAS transgene, which means that its expression is permanent and not transient, in contrast to what was observed under wild-type injury conditions (Ayaz, 2008).
Regeneration and tissue repair allow damaged or lost body parts to be replaced. After injury or fragmentation of Drosophila imaginal discs, regeneration leads to the development of normal adult structures. This process is likely to involve a combination of cell rearrangement and compensatory proliferation. However, the detailed mechanisms underlying these processes are poorly understood. A system was established to allow temporally restricted induction of cell death in situ. Using Gal4/Gal80 and UAS-rpr constructs, targeted ablation of a region of the disc could be performed and regeneration monitored without the requirement for microsurgical manipulation. Using a ptc-Gal4 construct to drive rpr expression in the wing disc resulted in a stripe of dead cells in the anterior compartment flanking the anteroposterior boundary, whereas a sal-Gal4 driver generated a dead domain that includes both anterior and posterior cells. Under these conditions, regenerated tissues were derived from the damaged compartment, suggesting that compartment restrictions are preserved during regeneration. These studies reveal that during regeneration the live cells bordering the domain in which cell death was induced first display cytoskeletal reorganisation and apical-to-basal closure of the epithelium. Then, proliferation begins locally in the vicinity of the wound and later more extensively in the affected compartment. Finally, regeneration of genetically ablated tissue was shown to require JNK activity. During cell death-induced regeneration, the JNK pathway is activated at the leading edges of healing tissue and not in the apoptotic cells, and is required for the regulation of healing and regenerative growth (Bergantiños, 2010).
Two main conclusions can be drawn from this work: (1) that genetically induced regeneration entails compartment-specific proliferation; and (2), that this type of regeneration requires JNK signalling for early regeneration events (Bergantiños, 2010).
This study established that the proliferation response to ptc>rpr induction is concentrated in the A compartment and consists of two activities: a local and a compartment-associated response. The local proliferation response resembles the activity of blastemas, a feature found in discs after fragmentation and implantation. The late compartment-restricted proliferation could be indicative of a reutilisation of developmental programs. The entire A compartment responds to the lack of the original ptc region by reactivating proliferation in order to achieve the final organ size. Thus, it is concluded that genetically induced regenerating discs restore the overall organ size by activation of proliferation, not only near the wound, as in fragmented and implanted discs, but also in the whole affected compartment. Thus, it is believed that the local proliferation is a fast and early response to the lost structures and that the later compartment-associated proliferation is a response to adjust the size of the tissue (Bergantiños, 2010).
ptc and sal were selected because of the precise removal of cells and also because they enabled testing whether both A and P compartments are involved in regeneration. The results suggest that when the A compartment is damaged (ptc>rpr), the P compartment only responds to the injury by sealing the gap that separates it from the A compartment through the generation of F-actin-rich cell extensions. These are projected to anchor the extensions from the cells at the edge of the A compartment as they proceed towards recovery of the intact cell sheet. In this situation, the regenerated tissue is derived exclusively from the A compartment. By contrast, when cells from both the A and P compartments are killed (sal>rpr), proliferation increases in both compartments. The boundaries between compartments are rapidly re-established after injury and prevent cells from crossing into adjacent compartments. Thus, boundaries are respected and compartments act as units of growth during regeneration (Bergantiños, 2010).
Following genetic ablation driven by either the ptc or sal drivers, healing starts at the DV boundary and spreads laterally towards the proximal regions, which are the last to close the wound. Cells at the DV boundary are arrested in G1-S, through a mechanism based on Notch and wg signalling. These arrested cells are the first to respond to healing and drive the cytoskeletal machinery for tissue reorganisation. This is consistent with the idea that the requirements for cell proliferation and for cell shape changes that occur during normal fly and vertebrate development and wound repair place incompatible demands on the cytoskeletal machinery of the cell. Another issue to be considered is that the DV boundary is the first zone of closure for F-actin extensions. This is reminiscent of Drosophila embryonic dorsal closure and wound repair, in which matching filopodia on both sides of the opening are recognised by the code of segment polarity genes in each parasegment. In addition, mechanical forces may be involved in tissue reorganisation. Stretching forces could be altered upon the induction of cell death, and they could have an important role in mounting a quick healing response. For example, mechanical forces, which have been proposed to act in the developing wing disc and compress the tissue through the central region, could stretch it towards the DV border after ablation of the ptc domain. Thus, either by matching affinities or by stretching forces, wound repair spreads from the apical DV border to basal and proximal domains (Bergantiños, 2010).
It has been shown in the Drosophila wing disc that massive loss of cells after irradiation gives rise to apparently normal adult wings as a result of compensatory proliferation driven by surviving cells. Experiments involving irradiation or induction of apoptosis in a p35 background have suggested that this compensatory proliferation is controlled by signals, including JNK, emerging from cells that have entered apoptosis, and that cell-death regulators, such as p53 and the caspase Dronc (Nedd2-like caspase), function as regulators of compensatory proliferation and blastema formation in the surviving cells. By contrast, the results show that proliferation is compartment specific and occurs independently of the dead tissue following targeted ablation. Two observations strongly support this interpretation. First, puc expression, as a marker of JNK activity, is concentrated in a narrow strip of apical cells, suggesting that JNK signalling is activated in the leading edges during wound closure. This again resembles other repair mechanisms described not only in imaginal discs, but also in other healing tissues, and reiterates epithelial fusion events observed in embryogenesis. Second, perturbation of the JNK pathway within the dying domain has no effect on either healing or regeneration. Even the early peak of localised mitosis near the wound and the later A-compartment-associated mitoses are present when UAS-bskDN and UAS-puc are driven in the dying domain. Effects on healing and regeneration are found only in hep mutant backgrounds, when JNK is impaired in the whole epithelium and not only in the dead domain. This requirement for the JNK pathway at the edges of the wound has also been found in studies of microsurgically induced regeneration. Cell lineage analysis of puc-expressing cells near the wound has shown that puc sets the limits of a blastema and that puc derivatives are able to reconstitute most of the missing tissue (Bergantiños, 2010).
Finally, whether JNK is required for healing alone or also functions as a signal for proliferation remains an open issue. Rapid local proliferation is affected in unhealed hep heterozygotes. Also, salPE>rpr wing regeneration cannot be achieved after 10 hours induction in a hepr75 background. Reduced proliferation could be due to a lack of healing or to loss of JNK activity. The possibility canot be ruled out that the JNK cascade, through the active AP-1 (Kayak and Jun-related antigen -- FlyBase) transcription factor complex, targets not only genes required for healing and epithelial fusion, but also those required for regenerative growth. In mammals, inhibition of the JNK pathway or lack of c-Jun results in eyelid-closure defects and also impairs proliferation by targeting Egfr transcription. Reconstruction of normal pattern and size might also require multiple signals. It has recently been found that regenerative growth induced by cell death requires Wnt/Wg signalling to increase dMyc stability, suggesting the involvement of other signalling pathways and also cell competition. It is very likely that an integrated network of signals and cell behaviours is necessary to reconstitute the damaged tissue (Bergantiños, 2010).
Taken together, these results suggest a model for cell-induced regeneration that includes two phases. The first, which occurs near the wound edges, involves JNK activity and is important for healing and rapid local proliferation. The second involves proliferation to compensate for the lost tissue and is extended throughout the damaged compartment. As in normal development, the regenerative growth that occurs in this second phase requires the reconstitution of morphogenetic signals that drive proliferation (Bergantiños, 2010).
The wound healing response is an essential mechanism to maintain the integrity of epithelia and protect all organisms from the surrounding milieu. In the 'purse-string' mechanism of wound closure, an injured epithelial sheet cinches its hole closed via an intercellular contractile actomyosin cable. This process is conserved across species and utilized by both embryonic as well as adult tissues, but remains poorly understood at the cellular level. In an effort to identify new players involved in purse-string wound closure a wounding strategy suitable for screening large numbers of Drosophila embryos was developed. Using this methodology, wound healing defects were observed in Jun-related antigen (encoding DJUN) and scab (encoding Drosophila alphaPS3 integrin) mutants and a forward genetics screen was performed on the basis of insertional mutagenesis by transposons that led to the identification of 30 lethal insertional mutants with defects in embryonic epithelia repair. One of the mutants identified is an insertion in the karst locus, which encodes Drosophila betaHeavy-spectrin. betaHeavy-spectrin (betaH) localizes to the wound edges where it presumably exerts an essential function to bring the wound to normal closure (Campos, 2010).
Using previously described DC or wound healing mutants a pilot screen was performed to validate the embryonic wounding strategy. The fact that a member of the DJNK pathway (Jra/DJun) was identified in the assay is in accordance with other reports that implicate this pathway in wound healing. Specifically, two mutations in components of the DJNK pathway, bsk/DJNK and kay/DFos, were previously shown to have defects in fly larval and adult wound closure, respectively. In addition, a reporter construct has been describes that requires consensus binding sites for the JUN/FOS complex to be activated upon wounding. Interestingly, treporter activation was still observed in Jra mutants, which suggests that additional signaling pathways are involved in wound closure (Campos, 2010).
An apparent discrepancy arose when the assay revealed a phenotype with Jra but not with puc mutants, another component of the same signaling pathway. This result might be explained by the fact that Jra and puc function in opposite directions in the DJNK signaling pathway. Puc functions as a pathway repressor, so in a puc mutant the JNK pathway should be less repressed and an opposite effect to a Jra mutation could be expected. In addition, activation of a puc-lacZ reporter has been shown to occur in larvae, wing imaginal discs, and adult wounds that take 18-24 hr to close, but it is only robustly detectable 4-6 hr postpuncture. Embryonic wounds are faster to heal, and even after inflicting a large laser wound on stage 14/15 embryos, no activation of the puc-lacZ reporter (assessed in open wounds 3 hr postwounding by immunofluorescence; data not shown) was detected. This observation suggests that, in rapidly healing epithelial wounds, the JNK pathway is not activated to high enough levels to trigger auto-inhibition (Campos, 2010).
The α-integrin scab was never before implicated in embryonic wound healing, but this mutant's phenotype comes as no great surprise. The first scab mutation was isolated due to its abnormal larval cuticle patterning. The scab gene encodes for Drosophila α-PS3 integrin, which is zygotically expressed in embryonic tissues undergoing invagination, tissue movement, and morphogenesis. Integrin proteins are involved in cell-matrix interactions and α-PS3 integrin regulation, in particular, mediates zipping of opposing epithelial sheets during DC. Similarly, the observation of a wound defect in scb5J38 mutants is consistent with a role for α-PS3 integrin in zipping of opposing epithelial cells during the healing process (Campos, 2010).
A previous study using confocal video microscopy has shown that Rho11B mutants take twice as long to close an epithelial wound when compared to wild type. Rho1 was confirmed in the assay to be important for wound healing, although with a weaker phenotype (22% of embryos had unclosed holes). This result shows nonetheless that the assay can be sensitive enough to pick up a 'weak' wound healing mutant such as Rho11B, which is still able to heal wounds albeit slower than wild type (Campos, 2010).
The genes identified in the screen represent a variety of functions indicating that wound healing is a complex mechanism that requires the participation of many cellular processes. A large class of the candidate mutants are involved in several aspects of gene expression, including factors that regulate chromatin remodeling (dUtx and Pc), elongation (dEaf), splicing (Glo and CG3294), and translation (CG33123). These factors are likely needed during wound healing for the induction of a repair transcriptome. Interestingly, JNK signaling-dependent Pc group (PcG) gene downregulation has been observed during imaginal disc regeneration. In addition, a recent study revealed that PcG methylases are downregulated during wound healing, while counteracting demethylases, Utx and Jmjd3, are upregulated. The results for the Pc and Utx mutants are consistent with these studies and highlight the importance of epigenetic reprogramming in the repair process (Campos, 2010).
Some of the genes such as arc-p20 and karst probably have a more direct role in the cell shape changes that drive the tissue morphogenetic movements during epithelial repair. The gene product of arc-p20 is a component of Arp2/3, a complex that controls the formation of actin filaments, and karst encodes a component of the spectrin membrane cytoskeleton. Also related to morphogenesis, CG12913 encodes an enzyme involved in the synthesis of chondroitin sulfate, which is usually found attached to proteins as part of a proteoglycan, suggesting a predictable contribution of the extracellular matrix in the tissue movements necessary for wound healing (Campos, 2010).
The epithelium is the first line of defense of the organism against pathogens and tissue integrity. It would thus seem plausible that genes involved in innate immunity could be identified with the screening protocol. Indeed, two of the genes (Ser12 and CG5198) seem to point to the involvement of the immune response in the healing of the laser-induced wounds. Ser12 is a member of the serine protease family, a class of proteins that has been shown to play a role in innate immunity. The CG5198 gene has no described function in Drosophila so far, but its homolog, CD2-binding protein 2, is involved in T lymphocyte activation and pre-RNA splicing. Another candidate that might represent a link to immunity is Atg2, a gene important for the regulation of autophagy, a process by which cells degrade cytoplasmic components in response to starvation. In Drosophila, autophagy has been linked to the control of cell growth, cell death, and, recently, to the innate immune response mechanism against vesicular stomatitis virus and listeria infection (Campos, 2010).
Isolation of an insertion in the stam gene points to the involvement of the JAK-STAT signaling cascade in this regenerative process. Interestingly, stam has been shown to be involved in Drosophila tracheal cell migration and is upregulated following Drosophila larvae infection by Pseudomonas entomophila (Campos, 2010).
One candidate could be involved in the uptake or export of some important wound signal (CG7627) as this gene encodes for a multidrug resistant protein (MRP), part of the ABC transporter superfamily, involved in drug exclusion properties of the Drosophila blood-brain barrier (Campos, 2010).
The kinase encoded by grapes is the Drosophila homolog of human Check1 (Chk1) involved in the DNA damage and mitotic spindle checkpoints. All the Chk1 literature has focused on its role during the cell cycle. However, the Drosophila late embryonic epithelium is a quiescent tissue, even after wounding. Understanding Grapes function in this context is a challenging task that could lead to new paradigms. One hypothesis is that Grapes is involved in tension sensing, as it is in the spindle checkpoint, or may uncover a cellular repair process that could help damaged cells 'decide' to either die by apoptosis or participate in the repair process (Campos, 2010).
The remaining genes with a putative function represent a wide range of general metabolic processes (aralar1, gs1-like, CG4389, CG9249, CG11089, and CG16833), suggesting that healing the epithelium is a highly demanding process (Campos, 2010).
Finally, a significant number of genes that have not yet been studied and do not contain identifiable protein domains (CG2813, CG31805, CG6005, CG6750, CG10217, CG15170, and CG30010) were selected. At the moment it is not possible to predict the role that these genes may play, but further study may help to identify novel wound healing regulatory mechanisms (Campos, 2010).
One of the mutants identified in the transposon screen was kstd11183, an insertion in the βH-spectrin locus. This mutation is likely producing a truncated protein terminating three amino acids into the P-element insertion. Other mutations identified in nearby segments 14 (kst14.1, kst2) and 16 (kst1) lead to the production of a detectable truncated protein so it is likely that karstd11183 mutation also gives rise to a truncated protein. These mutant forms of βH lack approximately half of the wild-type protein, including a COOH-terminal PH domain region, which is involved in targeting the protein to the membrane, thus producing a potential dominant negative form of βH. However, the karstd11183 mutant should still have maternally loaded wild-type protein, as previous studies describe a complete absence of maternal protein only by the third instar larval stage. This maternal contribution is likely the main reason that this mutant, as well as the other mutants isolated in the screen, does not have a fully penetrant wound healing phenotype (Campos, 2010).
βH-spectrin was shown to localize to the actomyosin purse string, a supracellular contractile cable that forms rapidly upon wound induction. Live imaging has demonstrated that actin and myosin can accumulate in this cable structure within minutes after wounding. Unfortunately, due to the size of the βH gene (>13 kb) cloning and tagging it for live imaging is not possible using standard methods, but the experiments in fixed tissue reveal that βH can accumulate very rapidly in this cable structure. βH accumulation was observed at the earliest time point technically feasible, 15 min postwounding. These observations are consistent with previous studies, also in fixed tissue, demonstrating rapid changes in βH localization during the process of cellularization in Drosophila embryos. Taken together, it is clear that at least the βH component of the membrane skeleton is not just a static structural scaffold as the name implies, but rather a dynamic protein capable of responding to or directing changes in cellular dynamics. The studies suggest that polarized redistribution of βH exerts an essential function to facilitate actin-based cellular responses, such as cable accumulation/maintenance and wound edge filopodia dynamics, which are necessary to properly close a wound (Campos, 2010).
βH has been previously observed in association with actin 'rings' during development of Drosophila and C. elegans. Arguably, C. elegans provides an example of actin ring function most analogous to the Drosophila wound edge purse string. During the final stages of C. elegans development, cortical arrays of actin in the outer epithelial cells, the hypodermis, dramatically reorganize to form parallel apically localized bundles of circumferencial supracellular actin rings. In this system, sma1, the C. elegans ortholog of βH, also localizes apically to these actin rings. In sma1 mutants the rings fail to productively contract and begin to disorganize, losing connection to the cell membranes. An additional phenotype observed in these mutants is the inability of cells to change their shape, a process normally 'directed' by these contractile rings, the end result being a short worm, a phenotype seen as functionally analogous to an unclosed wound in the Drosophila system (Campos, 2010).
In Drosophila, βH has been implicated in modulating cell shape changes during apical constriction of follicle cells (a process also involving actin rings) and has been proposed to function as a link between cross-linked actin networks/rings and the cell membrane. Further studies revealed that the C-terminal domain of βH has the ability to directly modulate the apical membrane area by regulating endocytosis, adding one more tantalizing piece of evidence pointing to the fact that βH could be a major player in cell shape changes, not only as a structural link but also by directly modulating the membrane area in response to cytoskeletal clues (or vice versa) (Campos, 2010).
Although it is known from previous studies that the actin cable is not absolutely required for wound closure, the process takes much longer without one. In Rho1 mutant embryos, cells lacking a cable are able to pull the wound closed using filopodia. The filopodial defect observed in karst mutants, adds another line of evidence to the absolute requirement of these structures for wound closure. In addition to the reduced actin cable accumulation and filopodial dynamics in karst mutants (which would lack the C-terminal domain responsible for membrane modulation), a lack of cell shape change is seen in the wound edge cells. Taken together, these data and the published work, introduce the intriguing possibility that βH could be serving as a link between wound edge dynamics and the coordinated cell shape changes usually observed in wild-type wound edge cells. The combination of the proposed ability of βH to modulate the apical membrane area as well as cross-link actin and act as an apical membranewide scaffold for other interactions, makes βH a good candidate to provide the physical link that would coordinate tissuewide actions, such as supracellular actin cable contraction, with the individual cellular responses, such as cell shape change and polarized filopodia activity (Campos, 2010).
Sleep is a unique behavioral state that is conserved between species, and sleep regulation is closely associated to metabolism and aging. Drosophila has been used to study the molecular mechanism underlying these physiological processes. This study shows that the c-Jun N-terminal Kinase (JNK) gene, known as basket (bsk) in Drosophila, functions in neurons to regulate both sleep and longevity in Drosophila. Pan-neuronal knockdown of JNK mRNA expression by RNA interference resulted in a decrease in both sleep and longevity. A heterozygous knockout of JNK showed similar effects, indicating the molecular specificity. The JNK knockdown showed a normal arousal threshold and sleep rebound, suggesting that the basic sleep mechanism was not affected. JNK is known to be involved in the insulin pathway, which regulates metabolism and longevity. A JNK knockdown in insulin-producing neurons in the pars intercerebralis had slight effects on sleep. However, knocking down JNK in the mushroom body had a significant effect on sleep. These data suggest a unique sleep regulating pathway for JNK (Takahama, 2011).
Recent studies have indicated that the MB is an important area for sleep regulation. Ablating the MB developmentally or inhibiting the MB neurons induces short sleep. Thus MB-Gal4 drivers, OK107 and eas-Gal4, were used to knockdown JNK, which resulted in a reduction in sleep. Both of these drivers cover the majority of neurons arborizing in the MB. In contrast, sleep was not affected significantly when JNK knockdown was induced in the PI by Dilp2-Gal4, where JNK signaling inversely regulates the production of insulin like peptide. Although there is a controversy over the relationship between diet-restriction induced longevity extension and insulin like peptide signaling, JNK has been reported to function in the PI to extend longevity. In these studies, JNK is supposed to function in the PI. This is in contrast to the function of JNK in sleep described in this study (Takahama, 2011).
Loss of metabolic homeostasis is a hallmark of aging and is commonly characterized by the deregulation of adaptive signaling interactions that coordinate energy metabolism with dietary changes. The mechanisms driving age-related changes in these adaptive responses remain unclear. This study characterized the deregulation of an adaptive metabolic response and the development of metabolic dysfunction in the aging intestine of Drosophila. Activation of the insulin-responsive transcription factor Foxo in intestinal enterocytes was found to be required to inhibit the expression of evolutionarily conserved lipases as part of a metabolic response to dietary changes. This adaptive mechanism becomes chronically activated in the aging intestine, mediated by changes in Jun-N-terminal kinase (JNK) signaling. Age-related chronic JNK/Foxo activation in enterocytes is deleterious, leading to sustained repression of intestinal lipase expression and the disruption of lipid homeostasis. Changes in the regulation of Foxo-mediated adaptive responses thus contribute to the age-associated breakdown of metabolic homeostasis (Karpac, 2013).
This work identifies Foxo-mediated repression of intestinal lipases as a critical component of an adaptive response to dietary changes in Drosophila. Interestingly, misregulation of this metabolic response also contributes to the age-associated breakdown of lipid homeostasis, as elevated JNK signaling leads to chronic Foxo activation and subsequent disruption of lipid metabolism due to chronic repression of lipases. This age-related deregulation of an adaptive metabolic response is reminiscent of insulin resistance-like phenotypes in vertebrates, which can also be triggered by chronic activation of JNK, and thus highlights the antagonistic pleiotropy inherent in metabolic regulation. The adaptive nature of signaling interactions that drive pathology (such as JNK-mediated insulin resistance) has remained elusive in many instances, and this work provides a model for age-related changes in an adaptive regulatory process that ultimately lead to a pathological outcome. It is believed that this system can serve as a productive model to address a number of interesting questions with relevance to the loss of metabolic homeostasis in aging organisms (Karpac, 2013).
In mammals, JNK has been shown to promote insulin resistance both cell-autonomously and systemically (through inflammation), subsequently affecting lipid homeostasis in various tissues. The current results further introduce a mechanism by which JNK can alter cellular and systemic lipid metabolism through the regulation of lipases, independent of changes in IIS. Thus, JNK-mediated Foxo activation in select tissues may be able to alter intracellular lipid metabolism, changing metabolic fuel substrates and disrupting metabolic homeostasis in other tissues without altering insulin responsiveness (Karpac, 2013).
Whereas the current data show that Foxo activation leads to the transcriptional repression of intestinal lipases, especially LipA/Magro, it remains unclear if this control is direct or indirect. Foxo is classically described as an activator of transcription, but recent reports have shown that Foxo can transcriptionally repress genes through direct association with promoters. The promoter regions of LipA/Magro and CG6295 do not contain conserved Foxo transcription factor binding sites, suggesting that the regulation of these genes may be indirect, potentially through Foxo-regulated expression of secondary effectors. Thus, tissue-specific control of lipid homeostasis by IIS/Foxo might be achieved through the regulation of lipogenic or lipolytic transcription factors that can elicit global and direct changes in cellular lipid metabolism. Previous reports have shown that the nuclear receptor dHR96, a critical regulator of lipid and cholesterol homeostasis, promotes lipA/magro expression. However, dhr96 expression is upregulated in aging intestines, suggesting that the age-related repression of intestinal lipases is not merely due to decreases in dHR96 levels. dhr96 transcript levels are strongly induced in genetic conditions where Foxo is activated and intestinal lipases are repressed, again suggesting that Foxo does not mediate its effects on lipase transcription by antagonizing dhr96 expression. Furthermore, age-related changes that are independent of JNK/Foxo activation may also contribute to the repression of intestinal lipase expression and disruption of lipid metabolism, such as an age-associated decline in feeding/food intake (Karpac, 2013).
The reasons for the increase in JNK and Foxo activity in aging enterocytes remain to be explored. Buchon (2009) has also shown that age-related activation of JNK in the intestinal epithelium is dependent on the presence of commensal bacteria, as maintaining animals axenically reduces activation of JNK in the first 30 days of life. Thus, bacteria-induced inflammation and subsequent JNK activation appears to be a likely cause, in part, for age-related increases in Foxo activity. In a separate study, however, this laboratory found that Foxo activation still occurs in intestines of old (40-day-old), axenically reared flies, suggesting that age-related activation of Foxo may also occur through JNK-independent processes. Supporting this idea, the results show that inhibiting JNK function in enterocytes can attenuate, although not completely inhibit, this Foxo activation. Additional factors, such as sirtuins or histone deacetylases, recently shown to deacetylate and activate Foxo in response to endocrine signals, may also lead to age-related increases in intestinal Foxo activity (Karpac, 2013).
Interactions between JNK and IIS/Foxo are mediated by various mechanisms. In mammals, JNK phosphorylates the insulin receptor substrate (IRS), inhibiting insulin signaling transduction. Whereas JNK has clearly been shown to antagonize IIS (activate Foxo) in C. elegans and Drosophila, that exact mechanism by which Foxo activation is achieved may be divergent in mammals. For example, no IRS homolog has been identified in worms, and the JNK phosphorylation site in mammalian IRS is not conserved in flies. The current data show that JNK-mediated Foxo activation in the aging fly intestine is not achieved through IIS antagonism upstream of Akt, suggesting either a direct interaction between Foxo and JNK or changes in other regulators of Foxo. Recent studies have shown that JNK-mediated phosphorylation of 14-3-3 proteins results in the release of their binding partners, including Foxo. The conservation of 14-3-3 proteins between vertebrates and invertebrates makes 14-3-3 an interesting candidate in promoting Foxo function via JNK in the aging fly intestine. This chronic intestinal Foxo activation and subsequent metabolic changes, provide a physiological system in Drosophila to genetically dissect the crosstalk between IIS/Foxo and JNK signaling. Detailed analysis of these signaling interactions promises to provide important insight into the pleiotropic effects of IIS/Foxo function and the pathogenesis of age-related metabolic diseases (Karpac, 2013).
The data further reveal the pleiotropic consequences of Foxo activation in regard to healthspan and longevity in Drosophila. Overexpressing Foxo in the fat body or muscle of flies leads to lifespan extension. The data presented here show that chronic Foxo activation in intestinal enterocytes disrupts lipid metabolism by deregulating intestinal lipases and thus highlight how cell- and tissue-specific consequences of Foxo function play an important role in determining either the beneficial (i.e., lifespan extension) or pathological (i.e., disruption of lipid metabolism) outcome of Foxo activation (Karpac, 2013).
Recent work in C. elegans has begun to explore the relationship between lipid metabolism and longevity, revealing that increases in intestinal lipase expression can extend lifespan. The beneficial effects of elevated lipase expression appear to be mediated by increases in specific types of fatty acids, which can activate autophagy and lead to lifespan extension. The current study identifies Foxo-mediated repression of intestinal lipases as a critical component of an adaptive response to dietary changes in Drosophila. Interestingly, misregulation of this metabolic response also contributes to the age-associated breakdown of lipid homeostasis, as elevated JNK signaling leads to chronic Foxo activation and subsequent disruption of lipid metabolism due to chronic repression of lipases. This age-related deregulation of an adaptive metabolic response is reminiscent of insulin resistance-like phenotypes in vertebrates, which can also be triggered by chronic activation of JNK, and thus highlights the antagonistic pleiotropy inherent in metabolic regulation. The adaptive nature of signaling interactions that drive pathology (such as JNK-mediated insulin resistance) has remained elusive in many instances, and the current work provides a model for age-related changes in an adaptive regulatory process that ultimately lead to a pathological outcome. This system can serve as a productive model to address a number of interesting questions with relevance to the loss of metabolic homeostasis in aging organisms (Karpac, 2013).
The results further introduce a mechanism by which JNK can alter cellular and systemic lipid metabolism through the regulation of lipases, independent of changes in IIS. Thus, JNK-mediated Foxo activation in select tissues may be able to alter intracellular lipid metabolism, changing metabolic fuel substrates and disrupting metabolic homeostasis in other tissues without altering insulin responsiveness (Karpac, 2013).
Whereas the data show that Foxo activation leads to the transcriptional repression of intestinal lipases, especially LipA/Magro, it remains unclear if this control is direct or indirect. Foxo is classically described as an activator of transcription, but recent reports have shown that Foxo can transcriptionally repress genes through direct association with promoters. The promoter regions of LipA/Magro and CG6295 do not contain conserved Foxo transcription factor binding sites, suggesting that the regulation of these genes may be indirect, potentially through Foxo-regulated expression of secondary effectors. Thus, tissue-specific control of lipid homeostasis by IIS/Foxo might be achieved through the regulation of lipogenic or lipolytic transcription factors that can elicit global and direct changes in cellular lipid metabolism. Previous reports have shown that the nuclear receptor dHR96, a critical regulator of lipid and cholesterol homeostasis, promotes lipA/magro expression. However, dhr96 expression is upregulated in aging intestines (data not shown), suggesting that the age-related repression of intestinal lipases is not merely due to decreases in dHR96 levels. dhr96 transcript levels are strongly induced in genetic conditions where Foxo is activated and intestinal lipases are repressed, again suggesting that Foxo does not mediate its effects on lipase transcription by antagonizing dhr96 expression. Furthermore, age-related changes that are independent of JNK/Foxo activation may also contribute to the repression of intestinal lipase expression and disruption of lipid metabolism, such as an age-associated decline in feeding/food intake (Karpac, 2013).
The reasons for the increase in JNK and Foxo activity in aging enterocytes remain to be explored. Age-related activation of JNK in the intestinal epithelium is dependent on the presence of commensal bacteria, as maintaining animals axenically reduces activation of JNK in the first 30 days of life. Thus, bacteria-induced inflammation and subsequent JNK activation appears to be a likely cause, in part, for age-related increases in Foxo activity. In a separate study, however, it was found that Foxo activation still occurs in intestines of old (40-day-old), axenically reared flies, suggesting that age-related activation of Foxo may also occur through JNK-independent processes. Supporting this idea, the current results show that inhibiting JNK function in enterocytes can attenuate, although not completely inhibit, this Foxo activation. Additional factors, such as sirtuins or histone deacetylases, recently shown to deacetylate and activate Foxo in response to endocrine signals, may also lead to age-related increases in intestinal Foxo activity (Karpac, 2013).
Interactions between JNK and IIS/Foxo are mediated by various mechanisms. In mammals, JNK phosphorylates the insulin receptor substrate (IRS), inhibiting insulin signaling transduction. JNK has also been shown to directly phosphorylate and activate Foxo in mammalian cell culture, that exact mechanism by which Foxo activation is achieved may be divergent in mammals. For example, no IRS homolog has been identified in worms, and the JNK phosphorylation site in mammalian IRS is not conserved in flies. The data show that JNK-mediated Foxo activation in the aging fly intestine is not achieved through IIS antagonism upstream of Akt, suggesting either a direct interaction between Foxo and JNK or changes in other regulators of Foxo. Recent studies have shown that JNK-mediated phosphorylation of 14-3-3 proteins results in the release of their binding partners, including Foxo. This chronic intestinal Foxo activation and subsequent metabolic changes, provide a physiological system in Drosophila to genetically dissect the crosstalk between IIS/Foxo and JNK signaling. Detailed analysis of these signaling interactions promises to provide important insight into the pleiotropic effects of IIS/Foxo function and the pathogenesis of age-related metabolic diseases (Karpac, 2013).
The data further reveal the pleiotropic consequences of Foxo activation in regard to healthspan and longevity in Drosophila. Overexpressing Foxo in the fat body or muscle of flies leads to lifespan extension. Overexpression of selected cytoprotective Foxo target genes in stem cells, on the other hand, is sufficient to prevent age-associated dysplasia and extend lifespan. The data presented here show that chronic Foxo activation in intestinal enterocytes disrupts lipid metabolism by deregulating intestinal lipases and thus highlight how cell- and tissue-specific consequences of Foxo function play an important role in determining either the beneficial (i.e., lifespan extension) or pathological (i.e., disruption of lipid metabolism) outcome of Foxo activation (Karpac, 2013).
Recent work in C. elegans has begun to explore the relationship between lipid metabolism and longevity, revealing that increases in intestinal lipase expression can extend lifespan. The beneficial effects of elevated lipase expression appear to be mediated by increases in specific types of fatty acids, which can activate autophagy and lead to lifespan extension. Interventions that promote lipid homeostasis with age, such as JNK/Foxo inhibition in intestinal enterocytes, might thus affect healthspan and/or longevity through means other than primarily maintaining energy homeostasis (Karpac, 2013).
The dedicator of cytokinesis (DOCK) family proteins that are conserved in a wide variety of species are known as DOCK1-DOCK11 in mammals. Sponge (Spg) is a Drosophila counterpart to the mammalian DOCK3. Specific knockdown of spg by pannier-GAL4 or apterous-GAL4 driver in wing discs induced split thorax phenotype in adults. Reduction of the Drosophila c-Jun N-terminal kinase (JNK), basket (bsk) gene dose enhanced the spg knockdown-induced phenotype. Conversely, overexpression of bsk suppressed the split thorax phenotype. Monitoring JNK activity in the wing imaginal discs by immunostaining with anti-phosphorylated JNK (anti-pJNK) antibody together with examination of lacZ expression in a puckered-lacZ enhancer trap line revealed the strong reduction of the JNK activity in the spg knockdown clones. This was further confirmed by Western immunoblot analysis of extracts from wing discs of spg knockdown fly with anti-pJNK antibody. Furthermore, the Duolink in situ Proximity Ligation Assay method detected interaction signals between Spg and Rac1 in the wing discs. Taken together, these results indicate Spg positively regulates JNK pathway that is required for thorax development and the regulation is mediated by interaction with Rac1 (Morishita, 2014PubMed).
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