The Interactive Fly

Zygotically transcribed genes

JNK pathway

  • Phosphorylation networks regulating JNK activity in diverse genetic backgrounds
  • Localized JNK signaling regulates organ size during development
  • The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila: Dying stem cells are engulfed by neighbouring differentiated cells through a JNK-dependent autophagy pathway
  • Spatiotemporal regulation of cell fusion by JNK and JAK/STAT signaling during Drosophila wound healing
  • ROS regulation of axonal mitochondrial transport is mediated by Ca2+ and JNK in Drosophila
  • Amyloid precursor like protein-1 promotes JNK-mediated cell migration in Drosophila
  • Signalling crosstalk at the leading edge controls tissue closure dynamics in the Drosophila embryo
  • GLYAT regulates JNK-mediated cell death in Drosophila
  • Feedback amplification loop drives malignant growth in epithelial tissues
  • Tankyrase regulates apoptosis by activating JNK signaling in Drosophila
  • Hippo signaling promotes Ets21c-dependent apical cell extrusion in the Drosophila wing disc
  • JNK and JAK/STAT signalling are required for inducing loss of cell fate specification during imaginal wing discs regeneration in Drosophila melanogaster
  • The ligand Sas and its receptor PTP10D drive tumour-suppressive cell competition
  • cdc37 is essential for JNK pathway activation and wound closure in Drosophila
  • The transcription factor Spalt and human homologue SALL4 induce cell invasion via the dMyc-JNK pathway in Drosophila
  • Novel function of N-acetyltransferase for microtubule stability and JNK signaling in Drosophila organ development
  • ZnT7 RNAi favors Raf(GOF)scrib(-/-)-induced tumor growth and invasion in Drosophila through JNK signaling pathway
  • Dpp and Hedgehog promote the glial response to neuronal apoptosis in the developing Drosophila visual system
  • Derlin-1 and TER94/VCP/p97 are required for intestinal homeostasis
  • Necrosis-induced apoptosis promotes regeneration in Drosophila wing imaginal discs
  • Anchor maintains gut homeostasis by restricting the JNK and Notch pathways in Drosophila
  • Distinct roles of Bendless in regulating FSC niche competition and daughter cell differentiation
  • Dissection of the Regulatory Elements of the Complex Expression Pattern of Puckered, a Dual-Specificity JNK Phosphatase
    Genes of JNK pathway Targets of the JNK pathway

    Phosphorylation networks regulating JNK activity in diverse genetic backgrounds

    Cellular signaling networks have evolved to enable swift and accurate responses, even in the face of genetic or environmental perturbation. Thus, genetic screens may not identify all the genes that regulate different biological processes. Moreover, although classical screening approaches have succeeded in providing parts lists of the essential components of signaling networks, they typically do not provide much insight into the hierarchical and functional relations that exist among these components. This study describes a high-throughput screen in which RNA interference was used to systematically inhibit two genes simultaneously in 17,724 combinations to identify regulators of Drosophila JUN NH2-terminal kinase (JNK). Using both genetic and phosphoproteomics data, an integrative network algorithm was then implemented to construct a JNK phosphorylation network, which provides structural and mechanistic insights into the systems architecture of JNK signaling (Bakal, 2008).

    Signaling networks, especially those maintaining cell viability and proliferation in response to environmental fluctuations and stress, may be more robust to perturbation than others. One signaling network dedicated to maintaining cell, tissue, and organism fidelity in the face of cellular stress involves stress-activated protein kinases (SAPKs), also known as JUN NH2-terminal kinases (JNKs). Classical in vivo genetic approaches in Drosophila have identified a highly conserved pathway consisting of a single JNK, a JNK-kinase (JNKK: Hemipterous), and a mixed-lineage kinase (MLK) that serves as a JNKK-kinase, but little is known as to how other signaling networks feed into this canonical cascade. To expand understanding of JNK regulation, cell-based RNA interference (RNAi) screens were conducted to systematically investigate JNK activity in various genetic backgrounds. Furthermore, to gain insight into the systems architecture of JNK signaling, a probabilistic computational framework was used to reconstruct a JNK phosphorylation network among components identified in the screen on the basis of phosphoproteomics data (Bakal, 2008).

    To measure JNK activity in live migratory Drosophila cells, an RNAi screen was devised based on a dJUN-FRET sensor (fluorescence resonance energy transfer or FRET). dJUN-FRET is a single polypeptide composed of a modified Drosophila JUN phosphorylation domain and a FHA phosphothreonine-binding module separated by a flexible linker and flanked by a cyan fluorescent protein (CFP) donor and yellow fluorescent protein (YFP) acceptor modules. Drosophila BG-2 migratory cells were transfected with a plasmid that drives dJUN-FRET expression from an actin promoter and, 2 days later, were transfected with a set of 1565 double-stranded RNAs (dsRNAs) targeting all 251 known Drosophila kinases, 86 phosphatases (PPases), and predicted kinases and PPases, as well as regulatory subunits and adapters (the 'KP' set). JNK activity in single cells was determined by calculating the ratio of FRET signal (generated by FRET between YFP and CFP) to the level of CFP intensity (which provides the baseline level of dJUN-FRET expression in each cell regardless of JNK activity) within each cell boundary. A mean ratio is then derived for all cells treated with a particular dsRNA. The mean fold change in dJUN-FRET reporter activity for 16,404 control wells was 1.00 ± 0.04 (SD); however, in a screen of the KP set, multiple dsRNAs targeting JNK (Z = -2.06 and -2.05) and MLK (Z = -5.06, -2.60, and -2.13) produced significant decreases in dJUN-FRET reporter activity. Moreover, dsRNAs targeting the JNK PPase puckered (puc) resulted in significant increases in reporter activity, consistent with the role of Puc as a negative regulator of JNK. In the KP screen, 24 genes (5% of genes tested) were identified as putative JNK regulators, and the 6 out of 7 positive and negative JNK regulators previously identified in vivo were reidentified. Although the KP screen identified both previously known and novel JNK components and regulators, the results are notable in the genes that the screen failed to isolate. For example, the only Drosophila JNKK, encoded by the hemipterous gene, was not identified in the KP screen. Furthermore, although ERK emerged from the KP screen as a JNK suppressor because of ERK's potential positive effects on puc transcription, no dsRNAs targeting other components of the ERK pathway were seen. A high false-negative rate appears to be present in this genetic screen; therefore, a combinatorial strategy was developed to further enhance the sensitivity of the screen (Bakal, 2008).

    Twelve different sensitized screens were developed in which cells were incubated with dsRNAs targeting a 'query' gene in combination with dsRNAs of the KP set. In choosing query genes, focus was placed primarily on components of Rho guanosine triphosphatase (GTPase) signaling, such as Rac1, Cdc42, the Rho guanine nucleotide exchange factor still-life (sif), and p190RhoGAP (GTPase-activating protein), because Rho activity couples JNK activation to a number of upstream signaling events. Cells were also sensitized by targeting canonical JNK components, such as JNK, puc, and MLK; other strong candidates from the KP screen, such as ERK; and genes, such as AKT, PTEN, hippo, and VHL, whose inhibition could result in the activation stress pathways even though they were themselves not identified in the KP screen. Genes were then identified as likely JNK regulators if two or more independent dsRNAs resulted in average increases or decreases in dJUN-FRET reporter activity in each screen, and a significance score was assigned based on how many total dsRNAs were tested for each gene across all screens. For example, a gene targeted by two to four dsRNAs was considered a JNK regulator if isolated in two or more screens, but a gene targeted by five to seven dsRNAs must be isolated in three or more screens to be included in the list of high-confidence regulators. No genes were isolated in the background of JNK inhibition, which showed that increases or decreases in dJUN-FRET reporter activity in both unmodified and modified backgrounds are JNK-dependent. Using this combinatorial approach, 55 new JNK suppressors and enhancers were identified in a test of 17,724 dsRNA combinations, which, together with results from the nonsensitized initial screen, provide a list of 79 likely JNK regulators (17% of the genes tested). Some of the hits identified in multiple screens were validated as bona fide JNK regulators by quantifying mRNA abundance of the JNK-specific transcriptional target MMP1 after dsRNA-mediated inhibition of candidate genes by quantitative real-time polymerase chain reaction (Bakal, 2008).

    Why do depletion of certain kinases and PPases had effects in both unmodified and modified backgrounds, while others were isolated only in sensitized contexts. To answer this question the genetic screen was integrated with phosphoproteomics data and computational models of kinase specificity to derive networks on the basis of all of these experimental sources using the NetworKIN algorithm. NetworKIN was deployed on more than 10,000 unique high-confidence phosphorylation sites identified in a recent mass spectrometry study of Drosophila cells (Bodenmiller, 2007). This resulted in an initial network that was subsequently overlaid with the genetic hits in order to derive a model of the JNK phosphorylation network. Last, to determine which phosphorylation events make functional contributions to JNK signaling, data sets derived from combinatorial screens for epistatic interactions among kinases and substrates were examined and hierarchical clustering of mean Z scores for components of the JNK phosphorylation network were performed across several combinatorial RNAi screens to look for shared patterns of genetic interaction. Thus, through integrating genetic and phosphoproteomics data using a computational framework, a systems-level strategy was undertaken to describe the protein networks underlying genetic interactions (Bakal, 2008).

    JNK regulators identified in all screens could be broadly grouped into different classes on the basis of previously described biological functions and/or structural similarity of protein products. Specifically, a number of protein and lipid kinases involved in axon guidance and cell migration were identified, such as FER, Ptp69d, otk, thickveins, RET, wunen2, GSK3, PDK1, and JAK. Genes encoding components of apicobasal polarity complexes were identifed, such as ZO-1, Caki, Magi, and discs large 1 (dlg1), largely as JNK suppressors, which is consistent with in vivo studies demonstrating unrestrained JNK activation associated with breakdown of polarity in backgrounds of hyperactivated Ras/ERK signaling. Furthermore, the results implicate the Warts-Hippo complex as a potential link between JNK activity and the remodeling of cytoskeletal structures. NetworKIN predicts that Hippo-mediated activation of JNK can occur through phosphorylation of MLK and that Hippo is also a direct target for JNK, which suggests that a feedback loop exists between JNK and Warts-Hippo signaling. Notably, it is also predicted that Dlg1 is extensively phosphorylated by a number of kinases in the JNK network, including JNK itself. This suggests that JNK, and other kinases such as ERK and CDK2, can act upstream of Dlg1 to remodel or dismantle polarized cell-cell adhesion complexes, which, in turn, promote the morphological changes required to complete division, migration, or extrusion from tissue during apoptosis. Compelling support of this idea is provided by the fact that mammalian Dlg1 is regulated by phosphorylation, is a substrate of JNKs, and becomes highly phosphorylated during mitosis. These findings highlight the ability of integrated genetic and computational approaches to provide systems-level insight into the complex regulation of JNK activity (Bakal, 2008).

    In summary, this study demonstrates that combinatorial RNAi screening is a powerful strategy to reduce the false-negatives present in current screens and reveals functions for a large fraction of genes. Moreover, the data-integrative-powered approach unraveled both mechanistic and hierarchical associations of components in the JNK regulatory system and provides an invaluable starting point for understanding the genetic interactions and signaling networks that underpin various diseases (Bakal, 2008).

    Localized JNK signaling regulates organ size during development

    A fundamental question of biology is what determines organ size. Despite demonstrations that factors within organs determine their sizes, intrinsic size control mechanisms remain elusive. This study shows that Drosophila wing size is regulated by JNK signaling during development. JNK is active in a stripe along the center of developing wings, and modulating JNK signaling within this stripe changes organ size. This JNK stripe influences proliferation in a non-canonical, Jun-independent manner by inhibiting the Hippo pathway. Localized JNK activity is established by Hedgehog signaling, where Ci elevates dTRAF1 expression. As the dTRAF1 homolog, TRAF4, is amplified in numerous cancers, these findings provide a new mechanism for how the Hedgehog pathway could contribute to tumorigenesis, and, more importantly, provides a new strategy for cancer therapies. Finally, modulation of JNK signaling centers in developing antennae and legs changes their sizes, suggesting a more generalizable role for JNK signaling in developmental organ size control (Willsey, 2016).

    Two independently generated antibodies that recognize the phosphorylated, active form of JNK (pJNK) specifically label a stripe in the pouch of developing wildtype third instar wing discs. Importantly, localized pJNK staining is not detected in hemizygous JNKK mutant discs, in clones of JNKK mutant cells within the stripe, following over-expression of the JNK phosphatase puckered (puc), or following RNAi-mediated knockdown of bsk using two independent, functionally validated RNAi lines (Willsey, 2016).

    The stripe of localized pJNK staining appeared to be adjacent to the anterior-posterior (A/P) compartment boundary, a location known to play a key role in organizing wing growth, and a site of active Hedgehog (Hh) signaling. Indeed, pJNK co-localizes with the Hh target gene patched (ptc). Expression of the JNK phosphatase puc in these cells specifically abrogated pJNK staining, as did RNAi-mediated knockdown of bsk. Together, these data indicate that the detected pJNK signal reflects endogenous JNK signaling activity in the ptc domain, a region of great importance to growth control. Indeed, while at earlier developmental stages pJNK staining is detected in all wing pouch cells, the presence of a localized stripe of pJNK correlates with the time when the majority of wing disc growth occurs (1000 cells/disc at mid-L3 stage to 50,000 cells/disc at 20 hr after pupation, so it is hypothesized that localized pJNK plays a role in regulating growth (Willsey, 2016).

    Inhibition of JNK signaling in the posterior compartment previously led to the conclusion that JNK does not play a role in wing development. The discovery of an anterior stripe of JNK activity spurred a reexamination of the issue. Since bsk null mutant animals are embryonic lethal, JNK signaling was conditionally inhibited in three independent ways in the developing wing disc. JNK inhibition was achieved by RNAi-mediated knockdown of bsk (bskRNAi#1or2), by expression of JNK phosphatase (puc), or by expression of a dominant negative bsk (bskDN). These lines have been independently validated as JNK inhibitors. Inhibition of JNK in all wing blade cells (rotund-Gal4, rn-Gal4) or specifically in ptc-expressing cells (ptc-Gal4) resulted in smaller adult wings in all cases, up to 40% reduced in the strongest cases. Importantly, expression of a control transgene (UAS-GFP) did not affect wing size. This contribution of JNK signaling to size control is likely an underestimate, as the embryonic lethality of bsk mutations necessitates conditional, hypomorphic analysis. Nevertheless, hypomorphic hepr75/Y animals, while pupal lethal, also have smaller wing discs, as do animals with reduced JNK signaling due to bskDN expression. Importantly, total body size is not affected by inhibiting JNK in the wing. Even for the smallest wings generated (rn-Gal4, UAS-bskDN), total animal body size is not altered (Willsey, 2016).

    To test whether elevation of this signal can increase organ size, eiger (egr), a potent JNK activator, was expressed within the ptc domain (ptc-Gal4, UAS-egr). Despite induction of cell death as previously reporte and late larval lethality, a dramatic increase was observed in wing disc size without apparent duplications or changes in the shape of the disc. While changes in organ size could be due to changing developmental time, wing discs with elevated JNK signaling were already larger than controls assayed at the same time point. Similarly, inhibition of JNK did not shorten developmental time. Thus, changes in organ size by modulating JNK activity do not directly result from altering developmental time. Finally, the observed increase in organ size is not due to induction of apoptosis, as expression of the pro-apoptotic gene hid does not increase organ size. In contrast, it causes a decrease in wing size. Furthermore, co-expression of diap1 or p35 did not significantly affect the growth effect of egr expression, while the effect was dependent on Bsk activity (Willsey, 2016).

    In stark contrast to known developmental morphogens, no obvious defects were observed in wing venation pattern following JNK inhibition, suggesting that localized pJNK may control growth in a pattern formation-independent manner. Indeed, even a slight reduction in Dpp signaling results in dramatic wing vein patterning defects. Second, inhibiting Dpp signaling causes a reduction in wing size along the A-P axis, while JNK inhibition causes a global reduction. Furthermore, ectopic Dpp expression increases growth in the form of duplicated structures, while increased JNK signaling results in a global increase in size. Molecularly, it was confirmed that reducing Dpp signaling abolishes pSMAD staining, while quantitative data shows that inhibiting JNK signaling does not. Furthermore, it was also found that Dpp is not upstream of pJNK, as reduction in Dpp signaling does not affect pJNK. Together, the molecular data are consistent with the phenotypic results indicating that pJNK and Dpp are separate programs in regulating growth. Consistent with these findings it has been suggested that Dpp does not play a primary role in later larval wing growth control (Akiyama, 2015). Finally, it was found that inhibition of JNK does not affect EGFR signaling (pERK) and that inhibition of EGFR does not affect the establishment of pJNK (Willsey, 2016).

    A difference in size could be due to changes in cell size and/or number. Wings with reduced size due to JNK inhibition were examined and no changes in cell size or apoptosis were found, suggesting that pJNK controls organ size by regulating cell number. Consistently, the cell death inhibitor p35 was unable to rescue the decreased size following JNK inhibition. Indeed, inhibition of JNK signaling resulted in a decrease in proliferation, while elevation of JNK signaling in the ptc domain resulted in an increase in cell proliferation in the enlarged wing disc. Importantly, this increased proliferation is not restricted to the ptc domain, consistent with previous reports that JNK can promote proliferation non-autonomously (Willsey, 2016).

    To determine the mechanism by which pJNK controls organ size, canonical JNK signaling through its target Jun was considered. Interestingly, RNAi-mediated knockdown of jun in ptc cells does not change wing size, consistent with previous analysis of jun mutant clones in the wing disc. Furthermore, in agreement with this, a reporter of canonical JNK signaling downstream of jun (puc-lacZ) is not expressed in the pJNK stripe. Finally, knockdown of fos (kayak, kay) alone or with junRNAi did not affect wing size. Together, these data indicate that canonical JNK signaling through jun does not function in the pJNK stripe to regulate wing size (Willsey, 2016).

    In search of such a non-canonical mechanism of JNK-mediated size control, the Hippo pathway was considered. JNK signaling regulates the Hippo pathway to induce autonomous and non-autonomous proliferation during tumorigenesis and regeneration via activation of the transcriptional regulator Yorkie (Yki). Recently it has been shown that JNK activates Yki via direct phosphorylation of Jub. To test whether this link between JNK and Jub could account for the role of localized pJNK in organ size control during development, RNAi-mediated knockdown of jub was performed in the ptc stripe, and adults with smaller wings were observed. Indeed, the effect of JNK loss on wing size can be partially suppressed in a heterozygous lats mutant background and increasing downstream yki expression in all wing cells or just within the ptc domain can rescue wing size following JNK inhibition. These results suggest that pJNK controls Yki activity autonomously within the ptc stripe, leading to a global change in cell proliferation. This hypothesis predicts that the Yki activity level within the ptc stripe influences overall wing size. Consistently, inhibition of JNK in the ptc stripe translates to homogeneous changes in anterior and posterior wing growth. Similarly, overexpression or inhibition of Yki signaling in the ptc stripe also results in a global change in wing size (Willsey, 2016).

    It is important to note that the yki expression line used is wild-type Yki, which is still affected by JNK signaling. For this reason, the epistasis experiment was also performed with activated Yki, which is independent of JNK signaling. Expression of this activated Yki in the ptc stripe caused very large tumors and lethality. Importantly, inhibiting JNK in this context did not affect the formation of these tumors or the lethality. Furthermore, inhibiting both JNK and Yki together does not enhance the phenotype of Yki inhibition alone, further supporting the idea that Yki is epistatic to JNK, instead of acting in parallel processes (Willsey, 2016).

    Mutants of the Yki downstream target four-jointed (fj) have small wings with normal patterning, and fj is known to propagate Hippo signaling and affect proliferation non-autonomously. Although RNAi-mediated knockdown of fj in ptc cells does not cause an obvious change in wing size, it is sufficient to block the Yki-induced effect on increasing wing size . However, overexpression of fj also reduces wing size, which makes it not possible to test for a simple epistatic relationship. Overall, these data are consistent with the notion that localized pJNK regulates wing size not by Jun-dependent canonical JNK signaling, but rather by Jun-independent non-canonical JNK signaling involving the Hippo pathway (Willsey, 2016).

    While morphogens direct both patterning and growth of developing organs, a link between patterning molecules and growth control pathways has not been established. pJNK staining is coincident with ptc expression, suggesting it could be established by Hh signaling. During development, posterior Hh protein travels across the A/P boundary, leading to activation of the transcription factor Cubitus interruptus (Ci) in the stripe of anterior cells. To test whether localized activation of JNK is a consequence of Hh signaling through Ci, RNAi-mediated knockdown of ci was performed, and it was found that the pJNK stripe is eliminated. Consistently, adult wing size is globally reduced. In contrast, no change was observed in pJNK stripe staining following RNAi-mediated knockdown of dpp or EGFR. Expression of non-processable Ci leads to increased Hh signaling. Expression of this active Ci in ptc cells leads to an increase in pJNK signal and larger, well-patterned adult wings. The modest size increase shown for ptc>CiACT is likely due to the fact that higher expression of this transgene (at 25 ° C) leads to such large wings that pupae cannot emerge from their cases. For measuring wing size, this experiment was performed at a lower temperature so that the animals were still viable. Furthermore, inhibition of JNK in wings expressing active Ci blocks Ci's effects, and resulting wings are similar in size to JNK inhibition alone . Together, these data indicate that Hh signaling through Ci is responsible for establishing the pJNK stripe (Willsey, 2016).

    To determine the mechanism by which Ci activates the JNK pathway, transcriptional profiles of posterior and ptc domain cells isolated by FACS from third instar wing discs were compared. Of the total 12,676 unique genes represented on the microarray, 50.4% (6,397) are expressed in ptc domain cells, posterior cells, or both. Hh pathway genes known to be differentially expressed were identified. It was next asked whether any JNK pathway genes are differentially expressed, and and it was found that dTRAF1 expression is more than five-fold increased in ptc cells, while other JNK pathway members are not differentially expressed (Willsey, 2016).

    dTRAF1 is expressed along the A/P boundary and ectopic expression of dTRAF1 activates JNK signaling. Thus, positive regulation of dTRAF1 expression by Ci could establish a stripe of pJNK that regulates wing size. Indeed, Ci binding motifs were identified in the dTRAF1 gene, and a previous large-scale ChIP study confirms a Ci binding site within the dTRAF1 gene. Consistently, a reduction in Ci led to a 29% reduction in dTRAF1 expression in wing discs. Given that the reduction of dTRAF1 expression in the ptc stripe is buffered by Hh-independent dTRAF1 expression elsewhere in the disc, this 29% reduction is significant. Furthermore, inhibition of dTRAF1 by RNAi knockdown abolished pJNK staining. Finally, these animals have smaller wings without obvious pattern defects. Conversely, overexpression of dTRAF1 causes embryonic lethality, making it not possible to attempt to rescue a dTRAF1 overexpression wing phenotype by knockdown of bsk. Nevertheless, it has been shown that dTRAF1 function in the eye is Bsk-dependent. Finally, inhibition of dTRAF1 modulates the phenotype of activated Ci signaling. Together, these data reveal that the pJNK stripe in the developing wing is established by Hh signaling through Ci-mediated induction of dTRAF1 expression (Willsey, 2016).

    Finally, localized centers of pJNK activity were detected during the development of other imaginal discs including the eye/antenna and leg. Inhibition of localized JNK signaling during development caused a decrease in adult antenna size and leg size. Conversely, increasing JNK signaling during development resulted in pupal lethality; nevertheless, overall sizes of antenna and leg discs were increased. Together, these data indicate that localized JNK signaling regulates size in other organs in addition to the wing, suggesting a more universal effect of JNK on size control (Willsey, 2016).

    Intrinsic mechanisms of organ size control have long been proposed and sought after. This study reveals that in developing Drosophila tissues, localized, organ-specific centers of JNK signaling contribute to organ size in an activity level-dependent manner. Such a size control mechanism is qualitatively distinct from developmental morphogen mechanisms, which affect both patterning and growth. Aptly, this mechanism is still integrated in the overall framework of developmental regulation, as it is established in the wing by the Hh pathway. These data indicate that localized JNK signaling is activated by Ci-mediated induction of dTRAF1 expression. Furthermore,it is not canonical Jun-dependent JNK signaling, but rather non-canonical JNK signaling that regulates size, possibly through Jub-dependent regulation of Yki signaling, as described for regeneration. As the human dTRAF1 homolog, TRAF4, and Hippo components are amplified in numerous cancers, these findings provide a new mechanism for how the Hh pathway could contribute to tumorigenesis. More importantly, these findings offer a new strategy for potential cancer therapies, as reactivating Jun in Hh-driven tumors could lead tumor cells towards an apoptotic fate (Willsey, 2016).

    The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila: Dying stem cells are engulfed by neighbouring differentiated cells through a JNK-dependent autophagy pathway

    Cancer stem cells (CSCs) may be responsible for tumour dormancy, relapse and the eventual death of most cancer patients. In addition, these cells are usually resistant to cytotoxic conditions. However, very little is known about the biology behind this resistance to therapeutics. This study investigated stem-cell death in the digestive system of adult Drosophila melanogaster. It was found that knockdown of the coat protein complex I (COPI)-Arf79F (also known as Arf1) complex selectively kills normal and transformed stem cells through necrosis, by attenuating the lipolysis pathway, but spares differentiated cells. The dying stem cells are engulfed by neighbouring differentiated cells through a draper-myoblast city-Rac1-basket (also known as JNK)-dependent autophagy pathway. Furthermore, Arf1 inhibitors reduce CSCs in human cancer cell lines. Thus, normal or cancer stem cells may rely primarily on lipid reserves for energy, in such a way that blocking lipolysis starves them to death. This finding may lead to new therapies that could help to eliminate CSCs in human cancers (Singh, 2016)

    To investigate the molecular mechanism behind the resistance of CSCs to therapeutics, the death of stem cells with different degrees of quiescence was studied in the adult Drosophila digestive system, including intestinal stem cells (ISCs). Expression of the proapoptotic genes rpr and p53 effectively ablated differentiated cells but had little effect on stem cells (Singh, 2016).

    In mammals, treatment-resistant leukaemic stem cells (LSCs) can be eliminated by a two-step protocol involving initial activation by interferon-α (IFNα) or colony-stimulating factor (G-CSF), followed by targeted chemotherapy. In Drosophila, activation of the hopscotch (also known as JAK)-Stat92E signalling pathway induces hyperplastic stem cells, which are overproliferating, but retain their apico-basal polarity and differentiation ability. A slightly different two-step protocol was conducted in Drosophila stem cells by overexpressing the JAK-Stat92E pathway ligand unpaired (upd) and rpr together. The induction of upd + rpr using the temperature-sensitive (ts) mutant esg-Gal4 (esgts > upd + rpr effectively ablated all of the ISCs and RNSCs through apoptosis within four days. Consistent with this result, expressing a gain-of-function Raf mutant (Rafgof) also accelerated apoptotic cell death of hyperplastic ISCs (Singh, 2016).

    Expressing a constitutively active form of Ras oncogene at 85D (also known as RasV12) in RNSCs and the knockdown of Notch activity in ISCs can transform these cell types into CSC-like neoplastic stem cells, which were not only overproliferating, but also lost their apico-basal polarity and differentiation abilit. It ws found that expressing rpr in RasV12-transformed RNSCs or in ISCs expressing a dominant-negative form of Notch (NDN) caused the ablation of only a proportion of the transformed RNSCs and few transformed ISCs and it did not affect differentiated cells; substantial populations of the neoplastic stem cells remained even seven days after rpr induction (Singh, 2016).

    These results suggest that the activation of proliferation can accelerate the apoptotic cell death of hyperplastic stem cells, but that a proportion of actively proliferating neoplastic RNSCs and ISCs are resistant to apoptotic cell death. Neoplastic tumours in Drosophila are more similar to high-grade malignant human tumours than are the hyperplastic Drosophila tumours (Singh, 2016).

    Vesicle-mediated COPI and COPII are essential components of the trafficking machinery for vesicle transportation between the endoplasmic reticulum and the Golgi. In addition, the COPI complex regulates the transport of lipolysis enzymes to the surface of lipid droplets for lipid droplet usage. In a previous screen, it was found that knockdown of COPI components (including Arf79F, the Drosophila homologue of ADP-ribosylation factor 1 (Arf1)) rather than COPII components resulted in stem-cell death, suggesting that lipid-droplet usage (lipolysis) rather than the general trafficking machinery between the endoplasmic reticulum and Golgi is important for stem-cell survival (Singh, 2016)

    To further investigate the roles of these genes in stem cells, a recombined double Gal4 line of esg-Gal4 and wg-Gal4 was used to express genes in ISCs, RNSCs, and HISCs (esgts wgts > X). Knockdown of these genes using RNA interference (RNAi) in stem cells ablated most of the stem cells in 1 week. However, expressing Arf79FRNAi in enterocytes or in differentiated stellate cells in Malpighian tubules did not cause similar marked ablation. These results suggest that Arf79F knockdown selectively kills stem cells and not differentiated cells (Singh, 2016).

    It was also found that expressing Arf79FRNAi in RasV12-transformed RNSCs ablated almost all of the transformed stem cells. Similarly, expressing Arf79FRNAi in NDN-transformed ISCs ablated all of the cells within one week, but restored differentiated cells to close to their normal levels within one week (Singh, 2016).

    δ-COP- and γ-COP-mutant clones were generated using the mosaic analysis with a repressible cell marker (MARCM) technique, and it was found that the COPI complex cell-autonomously regulated stem cell survival. In summary, knockdown of the COPI-Arf79F complex effectively ablated normal and transformed stem cells but not differentiated enterocytes or stellate cells (Singh, 2016)

    In the RNAi screen acyl-CoA synthetase long-chain (ACSL), an enzyme in the Drosophila lipolysis-β-oxidation pathway, and bubblegum (bgm), a very long-chain fatty acid-CoA ligase, were also identified. RNAi-mediated knockdown of Acsl and bgm effectively killed ISCs and RNSCs, but killed HISCs less effectively. Expressing AcslRNAi in RasV12-transformed RNSCs also ablated almost all of the transformed RNSCs in one week (Singh, 2016).

    Brummer (bmm) is a triglyceride lipase, the Drosophila homologue of mammalian ATGL, the first enzyme in the lipolysis pathway. Scully (scu) is the Drosophila orthologue of hydroxy-acyl-CoA dehydrogenase, an enzyme in the β-oxidation pathway. Hepatocyte nuclear factor 4 (Hnf4) regulates the expression of several genes involved in lipid mobilization and β-oxidation. To determine whether the lipolysis-β-oxidation pathway is required for COPI-Arf79F-mediated stem cell survival, upstream activating sequence (UAS)-regulated constructs (UAS-bmm, UAS-Hnf4, and UAS-scu) were also expressed in stem cells that were depleted of Arf79F, β-COP, or ζ-COP. Overexpressing either scu or Hnf4 significantly attenuated the stem cell death caused by knockdown of the COPI-Arf79F complex. Expressing UAS-Hnf4 MARCM clones also rescued the stem cell death phenotype induced by γ-COP knockdown. However, bmm overexpression did not rescue the stem-cell death induced by Arf79F knockdown. Since there are several other triglyceride lipases in Drosophila in addition to bmm, another lipase may redundantly regulate the lipolysis pathway (Singh, 2016)

    To further investigate the function of lipolysis in stem cells, the expression of a lipolysis reporter (GAL4-dHFN4; UAS-nlacZ which consisted of hsp70-GAL4-dHNF4 combined with a UAS-nlacZ reporter gene was investigated. The flies were either cultured continuously at 29°C or heat-shocked for 30 min at 37°C, 12 h before dissection. Without heat shock, the reporter was expressed only in ISCs and RNSCs of mature adult flies, but not in enteroendocrine cells, enterocytes, quiescent HISCs or quiescent ISCs of freshly emerged young adult flies (less than 3 days old. Expressing δ-COPRNAi almost completely eliminated the reporter expression, suggesting that the reporter was specifically regulated by the COPI complex. After heat shock or when a constitutively active form of JAK (hopTum-l) was expressed, the reporter was strongly expressed in ISCs, RNSCs and HISCs, but not in enteroendocrine cells or enterocytes. These data suggest that COPI-complex-regulated lipolysis was active in stem cells, but not in differentiated cells, and that the absence of the reporter expression in quiescent HISCs at 29°C was probably owing to weak hsp70 promoter activity rather than to low lipolysis in these cells (Singh, 2006).

    Lipid storage was futher investigated, and it was found that the size and number of lipid droplets were markedly increased in stem cells after knockdown of Arf79F (Singh, 2016).

    Arf1 inhibitors (brefeldin A, golgicide A, secin H3, LM11 and LG8) and fatty-acid-oxidation (FAO) inhibitors (triacsin C, mildronate, etomoxir and enoximone) were used, and it was found that these inhibitors markedly reduced stem-cell tumours in Drosophila through the lipolysis pathway but had a negligible effect on normal stem cells (Singh, 2016)

    These data together suggest that the COPI-Arf1 complex regulates stem-cell survival through the lipolysis-β-oxidation pathway, and that knockdown of these genes blocks lipolysis but promotes lipid storage. Further, the transformed stem cells are more sensitive to Arf1 inhibitors and may be selectively eliminated by controlling the concentration of Arf1 inhibitors (Singh, 2016)

    These data suggest that neither caspase-mediated apoptosis nor autophagy-regulated cell death regulates the stem-cell death induced by the knockdown of components of the COPI-Arf79F complex. Therefore whether necrosis regulates the stem-cell death induced by knockdown of the COPI-Arf79F complex was investigated. Necrosis is characterized by early plasma membrane rupture, reactive oxygen species (ROS) accumulation and intracellular acidification. Propidium iodide detects necrotic cells with compromised membrane integrity, the oxidant-sensitive dye dihydroethidium (DHE) indicates cellular ROS levels and LysoTracker staining detects intracellular acidification. The membrane rupture phenotype was detected only in esg and the propidium iodide signal was observed only in ISCs from flies that had RNAi-induced knockdown of expression of COPI-Arf79F components, and not in cells from wild-type flies. In the esgts wgts > AcslRNAi flies, all of the ISCs and RNSCs were ablated after four days at 29°C, but a fraction of the HISCs remained, and these were also propidium iodide positive, indicating that the HISCs were dying slowly. This slowness may have been due to either a lower GAL4 (wg-Gal4) activity in these cells compared to ISCs and RNSCs (esg-Gal4) or quiescence of the HISCs. Furthermore, strong propidium iodide signals were detected in transformed ISCs from esgts > NDN + Arf79FRNAi but not esgts flies, indicating that the transformed stem cells were dying through necrosis (Singh, 2016)

    Similarly, DHE signals were detected only in ISCs from esgts > Arf79FRNAi flies, indicating that the dying ISCs had accumulated ROS and were intracellularly acidified. Overexpressing catalase (a ROS-chelating enzyme) rescued the stem-cell death specifically induced by the γ-COP mutant clone, and the ROS inhibitor NAC blocked the Arf1 inhibitor-induced death of RasV12-induced RNSC tumours. These data together suggest that knockdown of the COPI-Arf1 complex induced the death of stem cells or of transformed stem cells (RasV12-RNSCs, NDN-ISCs) through ROS-induced necrosis. Although ISCs, RNSCs, and HISCs exhibit different degrees of quiescence, they all rely on lipolysis for survival, suggesting that this is a general property of stem cells (Singh, 2016)

    Cases were noticed where the GFP-positive material of the dying ISCs was present within neighbouring enterocytes, suggesting that these enterocytes had engulfed dying ISCs (Singh, 2016)

    The JNK pathway, autophagy and engulfment genes are involved in the engulfment of dying cells. Therefore, whether these genes are required for COPI-Arf79F-regulated ISC death was investigated. The following was found: (1) ISC death activated JNK signalling and autophagy in neighbouring enterocytes; (2) knockdown of these genes in enterocytes but not in ISCs rescued ISC death to different degrees; (3) the drpr-mbc-Rac1-JNK pathway in enterocytes is not only necessary but also sufficient for ISC death; and (4) inhibitors of JNK and Rac1 could block Arf1-inhibitor-induced cell death of the RasV12-induced RNSC tumours. These data together suggest that the drpr-mbc-Rac1-JNK pathway in neighbouring differentiated cells controls the engulfment of dying or transformed stem cells (Singh, 2016)

    The finding that the COPI-Arf79F-lipolysis-β-oxidation pathway regulated transformed stem-cell survival in the fly led to an investigation of whether the pathway has a similar role in CSCs. WTwo Arf1 inhibitors (brefeldin A and golgicide A) and two FAO inhibitors (triascin C and etomoxir) were tested on human cancer cell lines, and it was found that the growth, tumoursphere formation and expression of tumour-initiating cell markers of the four cancer cell lines were significantly suppressed by these inhibitors, suggesting that these inhibitors suppress CSCs. In mouse xenografts of BSY-1 human breast cancer cells, a novel low-cytotoxicity Arf1-ArfGEF inhibitor called AMF-26 was reported to induce complete regression in vivo in five days. Together, this report and the current results suggest that inhibiting Arf1 activity or blocking the lipolysis pathway can kill CSCs and block tumour growth (Singh, 2016)

    Stem cells or CSCs are usually localized to a hypoxic storage niche, surrounded by a dense extracellular matrix, which may make them less accessible to sugar and amino acid nutrition from the body's circulatory system. Most normal cells rely on sugar and amino acids for their energy supply, with lipolysis playing only a minor role in their survival. The current results suggest that stem cells and CSCs are metabolically unique; they rely mainly on lipid reserves for their energy supply, and blocking COPI-Arf1-mediated lipolysis can starve them to death. It was further found that transformed stem cells were more sensitive than normal stem cells to Arf1 inhibitors. Thus, selectively blocking lipolysis may kill CSCs without severe side effects. Therefore, targeting the COPI-Arf1 complex or the lipolysis pathway may prove to be a well-tolerated, novel approach for eliminating CSCs (Singh, 2016)

    Spatiotemporal regulation of cell fusion by JNK and JAK/STAT signaling during Drosophila wound healing

    Cell-cell fusion should be tightly controlled, but the underlying mechanism is poorly understood. This study found that the JAK/STAT pathway suppressed cell fusion during wound healing and delimited the event to the vicinity of the wound in the Drosophila larval epidermis. In the absence of JAK/STAT signaling, a large syncytium containing 3-fold the number of nuclei observed in wild-type tissue formed in wounded epidermis. upd2 and upd3 were transcriptionally induced by wounding and were required for suppressing excess cell fusion. JNK was activated in the wound vicinity and activity peaked at approximately 8 h after injury, whereas JAK/STAT signaling was activated in an adjoining concentric ring and activity peaked at a later stage. Cell fusion occurred primarily in the wound vicinity, where JAK/STAT activation was suppressed by fusion-inducing JNK signaling. JAK/STAT signaling was both necessary and sufficient for the induction of βPS integrin expression, suggesting that the suppression of cell fusion was mediated at least in part by integrin protein (Lee, 2017).

    ROS regulation of axonal mitochondrial transport is mediated by Ca2+ and JNK in Drosophila

    Mitochondria perform critical functions including aerobic ATP production and calcium (Ca2+) homeostasis, but are also a major source of reactive oxygen species (ROS) production. To maintain cellular function and survival in neurons, mitochondria are transported along axons, and accumulate in regions with high demand for their functions. Oxidative stress and abnormal mitochondrial axonal transport are associated with neurodegenerative disorders. However, little is known about the connection between these two. Using the Drosophila third instar larval nervous system as the in vivo model, this study found that ROS inhibited mitochondrial axonal transport more specifically, primarily due to reduced flux and velocity, but did not affect transport of other organelles. To understand the mechanisms underlying these effects, Ca2+ levels were studied along with the JNK (c-Jun N-terminal Kinase) pathway, which have been shown to regulate mitochondrial transport and general fast axonal transport, respectively. Elevated ROS was found to increase Ca2+ levels, and experimental reduction of Ca2+ to physiological levels rescued ROS-induced defects in mitochondrial transport in primary neuron cell cultures. In addition, in vivo activation of the JNK pathway reduced mitochondrial flux and velocities, while JNK knockdown partially rescued ROS-induced defects in the anterograde direction. It is concluded that ROS have the capacity to regulate mitochondrial traffic, and that Ca2+ and JNK signaling play roles in mediating these effects. In addition to transport defects, ROS produces imbalances in mitochondrial fission-fusion and metabolic state, indicating that mitochondrial transport, fission-fusion steady state, and metabolic state are closely interrelated in the response to ROS (Liao, 2017).

    Amyloid precursor like protein-1 promotes JNK-mediated cell migration in Drosophila

    The amyloid precursor like protein-1 (APLP1) is a member of the amyloid precursor protein (APP) family in mammals. While many studies have been focused on the pathologic role of APP in Alzheimer's disease, the physiological functions of APLP1 have remained largely elusive. This study reports that ectopic expression of APLP1 in Drosophila induces cell migration, which is suppressed by the loss of JNK signaling and enhanced by the gain of JNK signaling. APLP1 activates JNK signaling through phosphorylation of JNK, which up-regulates the expression of matrix metalloproteinase MMP1 required for basement membrane degradation and promotes actin remodeling essential for cell migration. These data thus provide the first in vivo evidence for a cell-autonomous role of APLP1 protein in migration (Wang, 2017).

    Signalling crosstalk at the leading edge controls tissue closure dynamics in the Drosophila embryo

    During Dorsal closure (DC), JNK (JUN N-terminal Kinase) signalling controls leading edge (LE) differentiation generating local forces and cell shape changes essential for DC. The LE represents a key morphogenetic domain in which, in addition to JNK, a number of signalling pathways converge and interact (anterior/posterior -AP- determination; segmentation genes, such as Wingless; Decapentaplegic). To better characterize properties of the LE morphogenetic domain, this study sought new JNK target genes through a genomic approach: 25 were identified, of which eight are specifically expressed in the LE, similar to decapentaplegic or puckered. Quantitative in situ gene profiling of this new set of LE genes reveals complex patterning of the LE along the AP axis, involving a three-way interplay between the JNK pathway, segmentation and HOX genes. Patterning of the LE into discrete domains appears essential for coordination of tissue sealing dynamics. Loss of anterior or posterior HOX gene function leads to strongly delayed and asymmetric DC, due to incorrect zipping in their respective functional domains. Therefore, in addition to significantly increasing the number of JNK target genes identified so far, the results reveal that the LE is a highly heterogeneous morphogenetic organizer, sculpted through crosstalk between JNK, segmental and AP signalling. This fine-tuning regulatory mechanism is essential to coordinate morphogenesis and dynamics of tissue sealing (Rousset, 2017).

    This identification of several new JNK target genes during DC and analysis of their quantitative expression patterns uncovers the complex transcriptional response taking place in the LE morphogenetic domain. Results reveal an intricate regulatory network integrating multiple signalling layers. In this process, AP positional information and JNK signalling cooperate to generate a highly patterned, yet apparently smooth and regular LE. Mutant analysis shows that LE partitioning into discrete domains is important to control the coordination, and hence the dynamics of the whole closure process (Rousset, 2017).

    The LE is a major component of DC, being the site of JNK activity and actin cable assembly; it also provides an active boundary with the amnioserosa, driving epidermal spreading and seamless tissue sealing. Therefore, it is important to determine its morphogenetic and signalling features and how these are dynamically controlled. To this end, a new set of target genes was identified whose expression in the dorsal ectoderm is dependent on JNK activity during DC. Transcriptome analysis allowed identification of 1648 independent genes which are up- or down-regulated in JNK activated embryos. Filtering of this large set yielded a group of 194 genes whose expression was analysed by quantitative in situ hybridization under different genetic conditions. Transcriptional profiling unveiled 31 Drosophila JNK target genes, of which only a fraction were already known, including jra/jun, reaper, Zasp52 and scab. Amongst novel targets were also Scaf and Rab30 the roles of which during DC have previously been described. Two categories of JNK target genes were distinguished: genes that are specifically expressed in the LE and genes whose expression is more ubiquitous in the dorsal ectoderm. Genes belonging to the latter category may play a general role in the ectoderm under the control of different pathways, for example in the case of Rab30. In contrast, LE-specific genes likely play a specific role during DC, as is the case for puc, dpp and scaf. However, it is also possible that some of the new genes, despite being expressed in the embryo in a JNK-dependent manner, are not involved in DC. These target genes thus remain under the control of JNK, but are functionally ‘silent’ during DC. This behaviour is best illustrated by reaper, whose expression is JNK-dependent in the embryo, but which does not seem to have any function in the LE, acting only later during development or at the adult stage (Rousset, 2017).

    Surprisingly, quantitative analysis of LE-specific gene expression profiles showed a variety of previously uncharacterized expression patterns along the LE, with two levels of regulation, AP and segmental. These observations reveal a new property of the LE which appears highly patterned along the AP axis, contrasting with the homogenous and linear structure previously envisioned. In addition, the higher order regulation that emerges from these results provides every LE cell with its own identity through the cross-talk between JNK, AP and segmental information. Such cell-level patterning through signalling crosstalk is likely essential for coordination and robustness of closure as well as segment matching. In this view, recent work showed that Wg and JNK interact at the LE to control the formation of specific mixer cells at segment boundaries (Rousset, 2017).

    Previous work showed that, instead of acting independently, HOX and segmentation genes can be coupled to regulate target genes in the embryo. This study revealed an additional layer of regulation involving the 'morphogenetic' JNK signalling pathway. During DC, JNK acts as a tissue-specific switch whose activity can be regulated by HOX and segmentation pathways, providing positional information an 'onion-like' regulatory model allows for several levels of regulation/information to pile up in order to regulate individual cellular behaviours important for tissue morphogenesis. Each layer can act positively or negatively on LE target gene expression, generating a complex repertoire of regulatory pathways. Distinct categories of expression profiles were identified in this study through the analysis of individual target genes, with the likelihood of more gene-specific patterns to be discovered. For example, the same HOX gene (abd-A or Abd-B) can have activating or repressive activity according to the target gene, as is the case for the transcription factor En. Molecular functional characterization of cis-regulatory elements controlling LE gene expression will bring a more detailed view of how transcription factor complexes are formed, how specificity of DNA recognition is achieved and how activating or repressive activities are regulated to generate LE patterning (Rousset, 2017).

    scaf proves to be a remarkable case among the JNK target genes, showing the different levels of regulation that can be integrated into a single promoter. Not only is it strongly expressed in the LE in a JNK-dependent manner, but it is also regulated by both the segmentation gene en and the HOX genes. In particular scaf displays a transcriptional response induced by all the trunk HOX genes tested, being positively controlled by Scr, Antp, Ubx and Abd-B and negatively by abd-A. It can therefore be considered as a general HOX target gene, i.e. regulated by most Hox paralogs, as previously defined. Another example of a general target is the Drosophila gene optix, which is activated by the head HOX genes labial and Deformed (Dfd) and inhibited by the trunk HOX genes. Nonetheless the general HOX target genes do not represent the majority. A genomic analysis in the Drosophila embryo identified more than 1500 genes regulated by at least one of the six HOX paralogs tested (Dfd, Scr, Antp, Ubx, abd-A, Abd-B). Only 1.3% of these genes are regulated by the six paralogs and 1.5% by the five paralogs that were used in this study. Interestingly more than 40% of the ~1500 HOX target genes are also present in the JNK genomic data set that was obtained. This strong overlap well reflects the fact that the LE runs along most of the body AP axis encompassing the thorax and abdomen. More importantly, it also indicates that AP patterning plays a crucial role in the regulation of DC, as shown in this study (Rousset, 2017).

    Live imaging and mathematical modelling revealed asymmetries in the geometry and zipping process along the AP axis; these can be attributed to local constraints induced by head involution and apoptosis. Head involution is concomitant with DC and induces tension in the anterior part of the embryo, explaining why the DC phenotypes are almost exclusively observed in the anterior part, leading to the so-called 'anterior-open phenotype'. The exception to this rule is the experimental manipulation of the posterior zipping rate through localized laser ablation of the amnioserosa close to the canthus, which induces a strong delay of posterior closure. The results with the abd-A and Abd-B mutants show that posterior delay can also be obtained in genetically-perturbed embryos. However, while anterior zipping is slightly up-regulated when posterior zipping is laser-targeted, it was shown that the anterior speed of closure is diminished in the Abd-B embryo. Thus, compensatory mechanisms may only appear when tissue integrity is severely impaired. Apoptosis was also proposed to participate in the asymmetric properties of DC. Delamination of apoptotic cells in the anterior amnioserosa produces forces that are responsible for a higher rate of anterior zipping. However, the phenotype that was observed with the abd-A or Abd-B mutation cannot be attributed to defects in this mechanism, as the rate of apoptosis is already very low in the posterior amnioserosa. In summary, the data reveal a genetic control of zipping through precise transcriptional regulation in the LE. Overall, this work provides a framework for apprehending how the HOX selector genes and their cofactors collaborate with other signalling pathways to generate specific transcriptional responses allowing morphogenetic patterning and proper coordinated development (Rousset, 2017).

    GLYAT regulates JNK-mediated cell death in Drosophila

    Cell death is a fundamental progress that regulates cell number, tissue homeostasis and organ size in development. The c-Jun N-terminal kinase (JNK) pathway has been evolutionarily conserved from fly to human, and plays essential roles in regulating cell death. To characterize additional genes that regulate JNK signaling, a genetic screen was performed in Drosophila and dGLYAT (CG34010), a novel gene whose function was previously unknown, was identified as a modulator of JNK-mediated cell death. Loss of dGLYAT suppressed JNK activation and cell death triggered by over-expression of Eiger or Hemipterous, or depletion of puckered or lgl in development, suggesting dGLYAT regulates both ectopic and physiological functions of JNK pathway. Furthermore, loss of dGLYAT was shown to inhibit JNK-mediated ROS production, suggesting dGLYAT regulates multiple functions of JNK signaling in vivo (Ren, 2017).

    Feedback amplification loop drives malignant growth in epithelial tissues

    Interactions between cells bearing oncogenic mutations and the surrounding microenvironment, and cooperation between clonally distinct cell populations, can contribute to the growth and malignancy of epithelial tumors. The genetic techniques available in Drosophila have contributed to identify important roles of the TNF-alpha ligand Eiger and mitogenic molecules in mediating these interactions during the early steps of tumor formation. This study unraveled the existence of a tumor-intrinsic-and microenvironment-independent-self-reinforcement mechanism that drives tumor initiation and growth in an Eiger-independent manner. This mechanism relies on cell interactions between two functionally distinct cell populations, and evidence is presented that these cell populations are not necessarily genetically different. Tumor-specific and cell-autonomous activation of the tumorigenic JNK stress-activated pathway drives the expression of secreted signaling molecules and growth factors to delaminating cells, which nonautonomously promote proliferative growth of the partially transformed epithelial tissue. Evidence is presented that cross-feeding interactions between delaminating and nondelaminating cells increase each other's sizes and that these interactions can explain the unlimited growth potential of these tumors. These results will open avenues toward molecular understanding of those social cell interactions with a relevant function in tumor initiation in humans (Muzzopappa, 2017).

    Tankyrase regulates apoptosis by activating JNK signaling in Drosophila

    Programmed cell death (PCD), or apoptosis, plays essential roles in various cellular and developmental processes, and deregulation of apoptosis causes many diseases. Thus, regulation of apoptotic process is very important. Drosophila tankyrase (DTNKS) is an evolutionarily conserved protein with poly (ADP-ribose) polymerase activity. In mammalian cells, tankyrases (TNKSs) have been reported to regulate cell death. To determine whether DTNKS plays function in inducing apoptosis in in vivo development, this study used Drosophila as a model system and generate transgenic flies expressing DTNKS. Ectopic expression of DTNKS promotes caspase-dependent apoptosis and knockdown of DTNKS by RNAi dramatically alleviates apoptotic defect caused by ectopic expression of pro-apoptotic proteins hid or rpr during eye development. Moreover, the result shows that ectopic expression of DTNKS triggers the activation of c-Jun N-terminal kinase (JNK) signaling, which is required for DTNKS-mediated apoptosis. Taken together, these findings have identified the role of DTNKS in regulating apoptosis by activating JNK signaling in Drosophila (Feng, 2018).

    Hippo signaling promotes Ets21c-dependent apical cell extrusion in the Drosophila wing disc

    Cell extrusion is a crucial regulator of epithelial tissue development and homeostasis. Epithelial cells undergoing apoptosis, bearing pathological mutations or possessing developmental defects are actively extruded toward elimination. However, the molecular mechanisms of Drosophila epithelial cell extrusion are not fully understood. This study reports that activation of the conserved Hippo (Hpo) signaling pathway induces both apical and basal cell extrusion in the Drosophila wing disc epithelia. Canonical Yorkie targets Diap1, Myc and Cyclin E are not required for either apical or basal cell extrusion (ACE and BCE) induced by activation of this pathway. Another target gene, bantam, is only involved in basal cell extrusion, suggesting novel Hpo-regulated apical cell extrusion mechanisms. Using RNA-seq analysis, it was found that JNK signaling is activated in the extruding cells. Genetic evidence is provided that JNK signaling activation is both sufficient and necessary for Hpo-regulated cell extrusion. Furthermore, it was demonstrate that the ETS-domain transcription factor Ets21c, an ortholog of proto-oncogenes FLI1 and ERG, acts downstream of JNK signaling to mediate apical cell extrusion. These findings reveal a novel molecular link between Hpo signaling and cell extrusion (Ai, 2020).

    Cell extrusion plays an important role in epithelial homeostasis and development as well as in cancer cell metastasis. In Drosophila epithelia, BCE occurs during dorsal closure and epithelial-mesenchymal transition (EMT) as well as in apoptosis, whereas ACE occurs in tumor invasion and extrusion of apoptotic enterocytes in the Drosophila adult midgut. However, the molecular mechanisms underlying BCE and ACE in Drosophila epithelia are not well understood. The current results demonstrate that inappropriate Hpo-Yki-JNK signaling induces ACE and BCE in Drosophila wing disc epithelia. This study also shows that in the wing disc epithelia, ban acts downstream of Yki to regulate BCE and Ets21c acts downstream of JNK to regulate ACE (Ai, 2020).

    The Hpo pathway regulates tissue growth in Drosophila. It has been reported that ykiB5 mutant clones grow poorly in the wing and eye discs. Consistent with these reports, the current results showed small ykiRNAi and ykiB5 mutant clones. Cells with depleted yki expression are extruded either apically or basally from the epithelia independently of apoptosis, indicating that cell extrusion is one explanation for the low recovery rate of Yki-depleted clones. In the Drosophila wing disc, overexpression of hpo by MS1096-Gal4 and nub-Gal4 dramatically decreases adult wing size. Meanwhile, overexpression of wts by nub-Gal4 also reduces the wing size. When hpo and wts expression, using C765-Gal4, cells were intensively extruded to the lumen and the basal side of the epithelia. Therefore, in addition to the proliferation defect, cell extrusion is one reason for the reduced tissue size induced by Hpo pathway activation. Diap1 levels are decreased in the small yki mutant clones, and co-expression of Diap1 and ykiRNAi could not block ACE or BCE. These results indicate that Diap1 does not regulate cell extrusion downstream of Yki. Hpo, wts mutant and yki overexpression in clones confers on cells supercompetitive properties that can lead to elimination of surrounding wild-type cells. This suggests that cell competition could promote elimination of Yki-depleted clones. In the current results, however, elimination of Yki-depleted cells could be triggered autonomously, even when Yki was depleted in the whole wing pouch. Cells expressing low levels of Myc are extruded basally through cell competition. Expressing Myc alone is not sufficient to prevent the elimination of yki mutant cells. Consistently, overexpression of Myc could not block BCE induced by silenced yki, indicating that other factors regulate BCE downstream of Yki. ban could inhibit ykiRNAi-mediated BCE but not ACE. It is known that activated Hpo plays a role in cell migration. Cells with depleted yki expression migrated across the AP boundary and were extruded basally, and this cell migration was suppressed by ban. These results show that ban can suppress ykiRNAi-induced BCE in the Drosophila wing disc but does not regulate ykiRNAi-induced ACE (Ai, 2020).

    In vertebrate epithelia, cells dying through apoptosis or crowding stress are extruded apically into the lumen. The S1P-S1P2 pathway regulates both apoptosis-induced and apoptosis-independent ACE. The oncogenic KRASV12G mutation in MDCK (Madin-Darby canine kidney) epithelial cell monolayers can downregulate both S1P (sphingosine 1-phosphate) and its receptor S1P2 (also known as S1PR2) to promote basal extrusion. In Drosophila epithelia, the direction of apoptotic cell extrusion is reversed with most apoptotic cells undergoing BCE. Apoptosis-induced BCE is regulated by JNK signaling. One exception is in Drosophila adult midgut, where enterocytes are lost through apical extrusion. However, little is known about the mechanism of ACE in Drosophila epithelia (Ai, 2020).

    In Drosophila epithelia, apical extrusion of scrib mutant cells is mediated by the Slit-Robo2-Ena complex, reduced E-cadherin and elevated Sqh levels. In normal cells, slit, robo2 and ena overexpression only results in BCE when cell death is blocked. More importantly, in the RNA-seq results, expression of slit, robo2 and ena were not changed in the Yki-depleted Drosophila wing disc, which means Slit-Robo2-Ena does not associate with the Hpo pathway to regulate ACE. scrib mutant cells activate Jak-Stat signaling and undergo ACE in the 'tumor hotspot' located in the dorsal hinge region of the Drosophila wing disc. Moreover, ACE can precede M6-deficient RasV12 tumor invasion following elevation of Cno-RhoA-MyoII. RNA-seq results revealed that the expression of Jak-Stat pathway genes and RhoA (Rho1) were not altered, indicating that ACE can be regulated by novel signaling pathways (Ai, 2020).

    In Drosophila, the JNK signaling pathway is essential for regulating cell extrusion in phenomena including wound healing, cell competition, apoptosis and dorsal closure. JNK signaling mediates the role of Dpp and its downstream targets in cell survival regulation in the Drosophila wing. Cell extrusion and retraction toward the basal side of the wing epithelia induced by the lack of Dpp activity is independent of JNK. In one case of ectopic fold formation at the AP boundary of the Drosophila wing, loss of Omb activates both Yki and JNK signaling. In this case, JNK signaling induces the AP fold by cell shortening, and Yki signaling suppresses JNK-dependent apoptosis in the folded cells. During cell competition induced by Myc manipulation, JNK-dependent apoptosis mediates the death of 'loser' cells and their extrusion to the basal side of the epithelia. Apoptosis-induced BCE can be blocked by Diap1, which suppresses JNK-dependent apoptosis. Taken together, these results show that JNK signaling mediates or interacts with Yki signaling in a cellular context-dependent manner during the regulation of wing epithelial morphogenesis and apoptosis (Ai, 2020).

    JNK is required for the migration of Csk mutant cells across the AP boundary and for their extrusion to the basal side of the epithelia. puc encodes a JNK-specific phosphatase that provides feedback inhibition to specifically repress JNK activity. Expression of puc can prevent ptc>CskRNAi cells from spreading at the AP boundary. JNK activity is also needed for ykiRNAi cells to invade across the wing disc AP boundary, and co-expression of bskDN and ykiRNAi blocks this invasion. Consistent with the role of JNK in BCE regulation, blocking JNK signaling by bskDN expression prevented ykiRNAi cells from being extruded to the basal side of the wing epithelia. More importantly, this study found that JNK activation by hepCA was sufficient to induce BCE, independently of apoptosis. Furthermore, few JNK targets have been shown to regulate cell migration and BCE. An exception to this are caspases that function downstream of JNK, which can promote cell migration when activated at a mild level (Ai, 2020).

    In Drosophila eye imaginal discs, elevated JNK signaling in scrib mutant cells regulates both ACE and BCE. JNK and Robo2-Ena constitute a positive-feedback loop that promotes the apical and basal extrusion of scrib mutant cells through E-cadherin reduction. Meanwhile, in normal cells, p35 upregulation when Robo2 and Ena are overexpressed only induces BCE. The current results showed that blocking JNK signaling could suppress ACE induced by silenced yki. Meanwhile, activation of JNK by hepCA was sufficient to induce the extrusion of cells into the lumen. Cell debris may be trapped in the disc lumen when overexpressing hepCA. Apoptosis was suppressed by co-expressing p35, to confirm that the ACE observed was independent of cell death. Taken together, these results indicate that there are additional regulators downstream of JNK to mediate ACE in normal cells (Ai, 2020).

    E-twenty-six (ETS) family transcription factors have conserved functions in metazoans. These include apoptosis regulation, cell differentiation promotion, cell fate regulation and cellular senescence. Ets21c encodes a member of the ETS-domain transcription factor family and is the ortholog of the human proto-oncogenes FLI1 and ERG. In Drosophila eye imaginal discs, 30-fold increased Ets21c expression is induced by RasV12 and eiger, an activator of JNK. In the Drosophila adult midgut, Ets21c expression is increased when JNK is activated by the JNK kinase hep. Ets21c can also promote tumor growth downstream of the JNK pathway. These results have confirmed that Ets21c functions downstream of JNK. Indeed, this study showed that Ets21c-GFP level was elevated following JNK activation. Expression of Ets21cHA was sufficient to induce ACE and silencing of Ets21c was sufficient to rescue ykiRNAi-induced ACE in the wing discs. However, the mechanism through which yki regulates JNK-Ets21c remains to be determined (Ai, 2020).

    In Drosophila imaginal discs, ACE promotes polarity-impaired cells to grow into tumors. Therefore, it is possible that Ets21c can promote Hpo-Yki-JNK-related tumorigenesis by facilitating ACE in Drosophila. It is difficult to infer a putative pro-tumoral function of Et21c in mammals through its effect on ACE. ACE is rather associated with the elimination of tumor cells in mammals, whereas BCE is traditionally associated with higher invasive capacity. Yki/YAP gain-of-function promotes cancer cell invasion in non-small-cell lung cancer, neoplastic transformation, uveal melanoma and pancreatic cancer. Additionally, Yki/YAP loss of function helps tumor cells to escape from apoptosis in hematologic malignancies, including multiple myeloma, lymphoma and leukemia. Consistent with the latter role, Yki suppressed cell extrusion from the Drosophila wing epithelia by suppressing Ets21c. Therefore, the role of Ets21c in Hpo-Yki-related tumor models should be further examined (Ai, 2020).

    JNK and JAK/STAT signalling are required for inducing loss of cell fate specification during imaginal wing discs regeneration in Drosophila melanogaster

    The regenerative process after tissue damage relies on a variety of cellular responses that includes compensatory cell proliferation and cell fate re-specification. The identification of the signalling networks regulating these cellular events is a central question in regenerative biology. Tissue regeneration models in Drosophila have shown that two of the signals that play a fundamental role during the early stages of regeneration are the c-Jun N-terminal kinase (JNK) and JAK/STAT signalling pathways. These pathways have been shown to be required for controlling regenerative proliferation, however their contribution to the processes of cellular reprogramming and cell fate re-specification that take place during regeneration are largely unknown. This study presents evidence for a previously unrecognised function of the cooperative activities of JNK and JAK/STAT signalling pathways in inducing loss of cell fate specification in imaginal discs. Co-activation of these signalling pathways induces both the cell fate changes in injured areas, as well as in adjacent cells. This function relies on the activity of the Caspase initiator encoded by the gene dronc (Ahmed-de-Prado, 2018).

    The ligand Sas and its receptor PTP10D drive tumour-suppressive cell competition

    Normal epithelial cells often exert anti-tumour effects against nearby oncogenic cells. In the Drosophila imaginal epithelium, clones of oncogenic cells with loss-of-function mutations in the apico-basal polarity genes scribble or discs large are actively eliminated by cell competition when surrounded by wild-type cells. Although c-Jun N-terminal kinase (JNK) signalling plays a crucial role in this cell elimination, the initial event, which occurs at the interface between normal cells and polarity-deficient cells, has not previously been identified. Through a genetic screen in Drosophila, this study identifies the ligand Sas and the receptor-type tyrosine phosphatase PTP10D as the cell-surface ligand-receptor system that drives tumour-suppressive cell competition. At the interface between the wild-type 'winner' and the polarity-deficient 'loser' clones, winner cells relocalize Sas to the lateral cell surface, whereas loser cells relocalize PTP10D there. This leads to the trans-activation of Sas-PTP10D signalling in loser cells, which restrains EGFR signalling and thereby enables elevated JNK signalling in loser cells, triggering cell elimination. In the absence of Sas-PTP10D, elevated EGFR signalling in loser cells switches the role of JNK from pro-apoptotic to pro-proliferative by inactivating the Hippo pathway, thereby driving the overgrowth of polarity-deficient cells. These findings uncover the mechanism by which normal epithelial cells recognize oncogenic polarity-deficient neighbours to drive cell competition (Yamamoto, 2017).

    Normal epithelial cells possess an intrinsic tumour-suppression mechanism against oncogenic neighbours. For instance, in canine kidney cell cultures and zebrafish embryos, oncogenic cells that activate Ras or Src are eliminated from an epithelial monolayer when surrounded by normal cells. Similarly, in the Drosophila imaginal epithelium, oncogenic polarity-deficient cells mutant for scribble (scrib) or discs large (dlg1; hereafter dlg) are eliminated from the tissue when surrounded by wild-type cells. The removal of these surrounding wild-type cells abolishes cell elimination and allows scrib- loss-of-function mutant cells to overproliferate; this context-dependent cell elimination is therefore considered to be cell competition. Genetic studies in Drosophila have revealed that this tumour-suppressive cell competition is driven by JNK-dependent cell death, triggered by the Drosophila tumour necrosis factor (TNF) Eiger. However, the initial mechanism by which normal epithelial cells recognize nearby polarity-deficient cells to drive cell competition have remained unknown (Yamamoto, 2017).

    To explore the initial event, which occurs at the interface between normal cells and oncogenic polarity-deficient cells, an ethyl methanesulfonate (EMS)-based genetic screen was conducted in Drosophila for genes required for wild-type 'winners' to eliminate neighbouring polarity-deficient 'losers'. In the eye imaginal epithelium, clones of homozygous mutant scrib-/- are eliminated when surrounded by wild-type tissue. The elimination of scrib-/- clones is also evident in adult eyes. Using the FLP/FRT-mediated genetic mosaic technique, EMS-induced homozygous mutations were induced only in wild-type winners and screened for mutations that caused an elimination-defective (eld) phenotype in neighbouring scrib- losers. Among 7,490 mutant strains generated, four elimination-defective mutants (eld-4, eld-6, eld-7, and eld-8) that fell into the same lethal complementation group were generated. Clones of scrib- cells surrounded by eld-4 clones were no longer eliminated but instead grew robustly in the eye disc and survived into adult tissue, causing a characteristic melanization phenotype. Notably, clones of eld-4, eld-6, eld-7, or eld-8 cells showed neither a growth disadvantage of their own nor a suppressive effect on the growth of neighbouring wild-type tissue. Thus, the complementation group eld-4/6/7/8 possesses mutations in a gene required for elimination of neighbouring scrib- clones (Yamamoto, 2017).

    Using a series of chromosomal-deficiency lines and subsequent cDNA sequencing, a nonsense mutation in the coding region of the gene stranded at second (sas) was identified in the eld-4 mutant strain. Encoded by sas is a cell-surface ligand protein that has two extracellular domains-von Willebrand factor type C (VWC) and fibronectin type 3 (FN3) domains-as well as a transmembrane domain. Sas is required for proper axon guidance in the nervous system, but its physiological role in epithelia is unknown. Expression of Sas was indeed lost in eld-4 clones, but ectopic expression of Sas within eld-4 clones surrounding scrib-/- clones reversed the elimination-defective phenotype. Moreover, the knockdown of Sas in cells surrounding scrib-/- clones phenocopied the elimination-defective phenotype; a similar elimination-defective phenotype also occurred upon Sas knockdown in cells surrounding dlg-/- mutant eye-disc clones. These data reveal that the cell-surface ligand Sas is required for normal epithelial cells to eliminate neighbouring polarity-deficient cells (Yamamoto, 2017).

    Next, attempts were made to understand the mechanism by which Sas drives the elimination of nearby cells. Sas is normally localized at the apical surface of epithelial cells. Notably, however, this study found that Sas relocalized to the lateral cell surface specifically at the interface between wild-type and scrib-/- or dlg-/- clones. This relocalization of Sas at the clone interface was also observed between wild-type and scrib-/- sas-/- double-mutant clones, indicating that the Sas protein that accumulates at the clone interface is derived from surrounding wild-type cells (Yamamoto, 2017).

    The fact that normal epithelial cells relocalize Sas laterally to eliminate neighbouring oncogenic cells suggests that normal cells transmit a signal to these cells through a cell-surface receptor for Sas. Attempts were made to identify the Sas receptor expressed in polarity-deficient cells. It has been reported that PTP10D, a receptor-type tyrosine phosphatase (RPTP), interacts and functions with Sas during longitudinal axon guidance in the Drosophila nervous system and that Sas-PTP10D trans-signalling occurs through glial-neuronal communication. It was therefore assumed that PTP10D and/or other RPTPs were strong candidates for the Sas receptor in the imaginal epithelium. Given that two extracellular domains of Sas, VWC and FN3, can form homophilic interactions with the same domains of other proteins and that FN3 is a domain commonly shared by RPTPs, Thirty-two RNA interference (RNAi) fly strains were screened that target expression of Drosophila transmembrane proteins bearing either VWC or FN3 domains. Only one RNAi line targeting PTP10D phenocopied the severe elimination-defective and melanization phenotypes when expressed within scrib-/- or dlg-/- mutant clones. Like Sas, PTP10D was relocalized to the interface between scrib-/- and wild-type clones, whereas it normally localized at the apical surface of epithelial cells. This lateral accumulation of PTP10D was almost eliminated when PTP10D-RNAi was expressed within scrib-/- clones, indicating that the PTP10D accumulating at the clone interface derives from scrib-/- mutant cells. Furthermore, immunostaining analysis of scrib-/-sas-/- double-mutant clones indicated that Sas and PTP10D are localized adjacent to each other in neighbouring cells. Notably, the lateral relocalization of Sas and PTP10D at the clone interface was also observed for the neoplastic non-functional tumour-suppressor mutants vps25-/-, erupted-/-, or Rab5DN-expressing cells, all of which are eliminated as losers of cell competition when surrounded by wild-type cells; however, such relocalization was not observed for non-neoplastic polarity stardust-/- or crumbs-/- mutants. These data suggest that in response to the emergence of neoplastic polarity-deficient cells, adjacent normal cells relocalize Sas laterally whereas nearby polarity-deficient cells relocalize PTP10D laterally, thereby driving elimination of polarity-deficient cells through trans-activated Sas-PTP10D signalling (Yamamoto, 2017).

    Next the mechanism by which Sas-PTP10D signalling drives elimination of polarity-deficient cells was investigated. It has previously been shown that the activation of Eiger-JNK signalling in polarity-deficient cells is essential for their elimination. Therefore, a possible mechanism by which PTP10D knockdown in scrib-/- clones results in an elimination-defective phenotype is through inhibition of JNK signalling. However, JNK signalling was still strongly activated in scrib-/- clones expressing PTP10D-RNAi, as assessed by the JNK target MMP1. This indicates that loss of PTP10D drives one or more intracellular signalling events that cause an elimination-defective phenotype in the presence of JNK activation. A strong candidate for this signalling event is activation of Ras signalling, as JNK is converted from pro-apoptotic to pro-growth in the presence of Ras activation. Notably, it has been reported that PTP10D and its mammalian orthologue PTPRJ (also known as DEP1/CD148/SCC1/RPTPeta) negatively regulate epidermal growth factor receptor (EGFR) signalling by directly dephosphorylating the intracellular tyrosine kinase domain of EGFR. This study found that EGFR normally localizes apically in wild-type cells but relocalizes to the lateral surface together with PTP10D at the boundaries between scrib-/- and wild-type clones. More pertinently, EGFR-Ras signalling was strongly elevated in scrib-/- clones expressing PTP10D-RNAi, as assessed by downregulation of the transcription factor Capicua. Moreover, co-knockdown of EGFR and PTP10D in scrib-/- clones completely reversed the elimination-defective phenotype, with EGFR-RNAi alone having only a slight effect on the growth of normal tissue. Furthermore, expression of a constitutively active form of EGFR or Ras caused overgrowth of scrib-/- clones, while expression of dominant-negative form of Ras in scrib-/-PTP10D-RNAi clones strongly suppressed their growth. Thus, scrib clones in the absence of PTP10D signalling activate both JNK and Ras signalling and overgrow in a manner dependent on EGFR signalling. The co-activation of EGFR-Ras and Eiger-JNK signalling causes hyper-accumulation of intracellular F-actin, thereby inactivating the tumour-suppressor Hippo pathway. Inactivation of the Hippo pathway triggers nuclear translocation and activation of the downstream transcriptional co-activator Yorkie (Yki), which induces upregulation of various pro-growth and anti-apoptotic genes. Indeed, scrib-/- clones expressing PTP10D-RNAi strongly accumulated intracellular F-actin and showed strong upregulation of the Yki target gene expanded (ex), as well as an increased nuclear signal of Yki protein; however, scrib mutation alone only slightly upregulated F-actin and ex expression. Furthermore, inhibition of Yki activity by the Yki kinase Warts (Wts) or Yki-RNAi significantly suppressed growth of scrib-/- clones in the absence of PTP10D, while Wts-overexpression or Yki-RNAi alone had little effect on tissue growth. Similar upregulations of EGFR signalling and Yki activity were observed in scrib-/- clones when surrounded by sas-/- eld-4 clones. Finally, he number of dying cells at the boundaries between scrib-/- and wild-type clones was found to be significantly reduced by PTP10D-knockdown, whereas cell proliferation was significantly increased in scrib-/- clones expressing PTP10D-RNAi. Together, these data indicate that when neoplastic polarity-deficient cells emerge in the epithelium, neighbouring non-neoplastic cells restrain EGFR signalling of nearby polarity-deficient cells through a Sas-PTP10D trans-interaction, which enables JNK signalling activated in polarity-deficient cells to drive cell elimination. In the absence of Sas-PTP10D, elevated EGFR-Ras signalling in polarity-deficient cells cooperates with JNK signalling to cause Yki activation, thereby leading to an elimination defect and overgrowth of polarity-deficient cells (Yamamoto, 2017).

    These data indicate that in response to the emergence of oncogenic polarity-deficient cells, Sas and PTP10D relocalize specifically at the clone interface to the respective lateral surfaces of normal or polarity-deficient cells, enabling the ligand and receptor to interact with each other in trans. Thus, Sas-PTP10D acts as a fail-safe system for epithelial tissue, a system that protects against neoplastic development and is normally latent but activates upon oncogenic cell emergence. Notably, the Sas-PTP10D system was not required for other types of cell competition triggered by Minute, Mahjong, Myc or Yki. Although the mechanism by which Sas and PTP10D relocalize to the clone interface is currently unknown, this study found that the apical proteins Bazooka, Patj, and aPKC and the sub-apical protein E-cadherin also relocalize to the lateral surface of the clone boundary. This suggests that the apical cell surface expands to the lateral region at the clone boundary, meaning that Sas and PTP10D meet each other in trans at the clone interface (Yamamoto, 2017).

    The genetic data reveal that Sas and PTP10D act together as tumour suppressors during cell competition. Previous studies have reported that PTPRJ, the mammalian homologue of PTP10D, also acts as a tumour suppressor and negatively regulates EGFR signalling. Although no obvious homologues of Sas have been identified in mammals, thrombospondin-1 and syndecan-2 have been reported to act as ligands for PTPRJ. Given that elimination of scrib-deficient cells by cell competition also occurs in mammalian systems, and that the signalling mechanisms identified in Drosophila are evolutionarily conserved, similar cell-cell recognition mechanisms may help to safeguard human tissues against tumorigenesis (Yamamoto, 2017).

    cdc37 is essential for JNK pathway activation and wound closure in Drosophila

    Wound closure in the Drosophila larval epidermis mainly involves non-proliferative, endocyling epithelial cells. Consequently, it is largely mediated by cell growth and migration. Both cell growth and migration in Drosophila require the co-chaperone-encoding gene cdc37. Larvae lacking cdc37 in the epidermis failed to close wounds, and the cells of the epidermis failed to change cell shape and polarize. Likewise, wound-induced cell growth was significantly reduced, and correlated with a reduction in the size of the cell nucleus. The c-Jun N-terminal kinase (JNK) pathway, which is essential for wound closure, was not typically activated in injured cdc37 knockdown larvae. In addition, JNK, Hep, Mkk4, and Tak1 protein levels were reduced, consistent with previous reports showing that Cdc37 is important for the stability of various client kinases. Protein levels of the integrin beta subunit and its wound-induced protein expression were also reduced, reflecting the disruption of JNK activation, which is crucial for expression of integrin beta during wound closure. These results are consistent with a role of Cdc37 in maintaining the stability of the JNK pathway kinases, thus mediating cell growth and migration during Drosophila wound healing (Lee, 2019).

    The healing of a mammalian skin wound is complex and involves various cellular processes, including blood clotting, inflammation, epithelial cell proliferation and migration, and matrix synthesis and remodeling, which span multiple tissues. In contrast, wound healing in the Drosophila larval epidermis is simple: the epidermis consists mainly of a single, nonproliferative cell layer that underlies the protective cuticle. Thus, wound closure involves primarily cuticle regeneration and cell growth and migration, but not proliferation (Lee, 2019).

    Many signaling pathways are required for wound closure in the Drosophila epidermis. Of these, c-Jun N-terminal kinase (JNK), which is required for a broad range of wound healing processes, is the most crucial. Without JNK, cells cannot properly polarize, change shape, orient toward the wound center, or migrate to close the wound. Conversely, some proteins acting upstream of JNK appear to be redundant in a pathway that includes both canonical and noncanonical factors in regard to the embryonic dorsal closure process. Specifically, both JNK and the AP-1 transcription factors DJun (Jra) and DFos (Kay) are absolutely required for wound closure, and larvae that are lacking any one of these factors cannot repair open wounds. In contrast, the Jun/stress-activated protein (SAP) 2 kinases Hep and Mkk4 are partially redundant, as are the Jun/SAP3 kinases Slpr and Tak1 . Although the involvement of the JNK/SAPK pathway in wound healing is well known both in insects and mammals, the mechanisms underlying the regulation of this pathway are not well understood (Lee, 2019).

    Protein kinases are often associated with the molecular chaperone Hsp90, which helps these client proteins take on their active conformation. Hsp90 interacts with at least 20 other factors, called cochaperones, which either modulate the activity of Hsp90 or affect the specificity of Hsp90 client proteins. Cdc37 is one such cochaperone that is known to maintain the function and stability of client kinases, and many kinases are regulated by Cdc37, but the relationship between Cdc37 and the JNK signaling pathway is not clear (Lee, 2019). A speculative model for the Hsp90-Cdc37-client protein cycle has been suggested. Cdc37 first tests the proper substrates and establishes stable connection with the client protein to create a Cdc37-client protein binary complex. Then, the binary complex binds to Hsp90 to form a ternary complex. The formation of the Hsp90-Cdc37-client protein ternary complex finally facilitates client protein loading onto the Hsp90 chaperone machinery. It is worthy of note that Cdc37 will be phosphorylated by casein kinase 2 (CK2) at Ser13 before connecting with client proteins and dephosphorylated by the protein phosphatase 5 (PP5) before client protein release (Li, 2018)

    Cdc37 was originally identified as a yeast cell-cycle regulator that was later found to interact with Hsp90 and v-Src. Hsp90 and Cdc37 are both structurally and functionally conserved in metazoans. In Drosophila, cdc37 was initially isolated from a mutagenesis screen based on its involvement in eye development, and was later found to be essential for Sevenless receptor tyrosine kinase signaling. Null mutations in cdc37 are recessively lethal, indicating it is required for cell viability. Cdc37 inhibits Hh and Wnt signaling pathways in both flies and mammalian cells and mediates chromosome segregation and cytokinesis by modulating the function of Aurora B kinase (Lee, 2019).

    This study isolated cdc37 based on its RNA interference (RNAi) knockdown phenotype in larval epidermal wound closure in Drosophila, and found that cdc37 is required not only for reepithelialization but also for cells to change shape, polarize, and grow during epidermal wound closure, and all of these phenotypes are shared by larvae lacking JNK. Molecularly, cdc37 is required for maintaining the protein levels of JNK pathway components (Lee, 2019).

    In Drosophila, JNK mediates diverse wound healing responses, including gene expression, cell shape change and polarization, reepithelialization, and cell fusion. The present study suggests that the JNK pathway also mediates wound-induced endoreplication and cell growth. However, prior reports have indicated that JNK suppresses wound-induced endoreplication in adult stages, which is a discrepancy that requires further investigation (Lee, 2019).

    Larvae lacking cdc37 displayed disrupted activation of the JNK pathway and displayed phenotypes similar to those of larvae lacking active JNK. Thus, it is concluded that most of the cdc37-knockdown phenotypes analyzed were likely caused by the disruption of JNK activation during wound healing. It should be noted, however, that cell nucleus size and JNK protein levels were also reduced in the unwounded epidermis of cdc37 knockdown larvae, indicating that loss of cdc37 expression also causes developmental defects. This was not unexpected, given that cdc37 null mutations are cell lethal (Lange, 2002). Considering that A58-GAL4 is only active after early larval stages, and that endoreplicating cells are resistant to apoptosis, the wound healing defects in cdc37-knockdown larvae may have been uncovered luckily due to cell stress caused by wounding in the apoptosis-resistant epidermal cells (Lee, 2019).

    Cdc37 is best known as a cochaperone that confers client kinase specificity to Hsp90 (Karnitz, 2007; Taipale, 2010). The client kinases requiring the Hsp90-Cdc37 complex for activity and stability are diverse and include Cdk2, Cdk4, Src, Aurora B, Raf1, and RIP3. However, Cdc37 may also function as an independent molecular chaperone alone, similar to Hsp90. This investigation into the possible involvement of Hsp90 (also known as Hsp83 in Drosophila) in wound healing using multiple Hsp90 RNAi lines did not yield any definitive answer. This study also assessed whether aurora B, cdc2, or ckII were involved in wound healing, as these factors reportedly interact with cdc37 in various contexts. Larvae deficient of each of these factors closed wounds normally. Finally, no noticeable changes were found in the protein level or localization of Cdc37 during wound healing. Thus, the requirement of cdc37 for JNK activation is a novel finding. Nonetheless, defining the detailed molecular mechanisms underlying Cdc37 functions requires further investigation (Lee, 2019).

    The transcription factor Spalt and human homologue SALL4 induce cell invasion via the dMyc-JNK pathway in Drosophila

    Cancer cell metastasis is a leading cause of mortality in cancer patients. Therefore, revealing the molecular mechanism of cancer cell invasion is of great significance for the treatment of cancer. In human patients, the hyperactivity of transcription factor Spalt-like 4 (SALL4) is sufficient to induce malignant tumorigenesis and metastasis. This study found that when ectopically expressing the Drosophila homologue spalt (sal) or human SALL4 in Drosophila, epithelial cells delaminated basally with penetration of the basal lamina and degradation of the extracellular matrix, which are essential properties of cell invasion. Further assay found that sal/SALL4 promoted cell invasion via dMyc-JNK signaling. Inhibition of the c-Jun N-terminal kinase (JNK) signaling pathway through suppressing matrix metalloprotease 1 or basket can achieve suppression of cell invasion. Moreover, expression of dMyc, a suppressor of JNK signaling, dramatically blocked cell invasion induced by sal/SALL4 in the wing disc. These findings reveal a conserved role of sal/SALL4 in invasive cell movement and link the crucial mediator of tumor invasion, the JNK pathway, to SALL4-mediated cancer progression (Sun, 2020).

    Human SALL4 has been reported to be significantly elevated in metastatic cancer cells. This study provides genetic evidence for a model in which sal/SALL4 regulates cell invasiveness by dMyc-JNK signaling. The JNK pathway is an important cellular signaling pathway that regulates a variety of cellular activities relevant to tumorigenesis, such as cell migration, apoptosis and proliferation. JNK promotes the expression of Mmp1, which acts as an enzyme to degrade basement membrane and ECM components to promote tumor cell motility. Manipulation of expression of many genes can lead to cell death, cell extrusion and invasive cell migration through activation of JNK signaling. sal/SALL4 overexpression activates Mmp1 and reducing JNK can suppress cell invasion and Mmp1 level. In addition to Mmp1, some other markers in the JNK pathway such as pJNK (activated bsk) and puc showed a significant increase in expression. Promotion of cell invasion by sal/SALL4 induction was accompanied by activation of the apoptotic pathway, but it was not dependent on apoptosis because caspase inhibition did not prevent cell invasion upon sal/SALL4 expression. Therefore, the JNK pathway probably mediates the role of sal/SALL4 overexpression to regulate cell invasion through an apoptosis-independent mechanism (Sun, 2020).

    The MYC gene is one of the most highly amplified oncogenes among many human cancers. For instance, in some certain cancer cells, Myc is upregulated through directly transcriptional activation by SALL4. Besides promoting cancer progression and metastasis, MYC has a bivalent role in regulating tumorigenesis and cell invasion. MYC restrains breast cancer cell motility and invasion through transcriptional silencing of integrin subunits. In Drosophila, dMyc inhibits JNK signaling in retinal progenitors to block non-autonomous glia over-migration (Tavares, 2017). The Drosophila puc gene, encoding the sole JNK-specific MAPK phosphatase and inhibitor, and its mammalian homologue Dusp10 are directly bound by Myc as shown in ChIP-sequencing data . In Drosophila tissues, direct evidence illustrates that dMyc and cMyc activate puc transcription through binding to the Myc binding-motif EB3, and consequently inhibit JNK signaling to suppress cell invasion. This study found that dMyc is repressed in sal/SALL4-expressing regions and introducing dMyc partially rescues cell invasion, indicating a repressive role of dMyc in tumor cell migration. As Sal is a transcriptional repressor in both Drosophila and human cells, it is possible that Sal/SALL4 binds to Myc and suppresses its expression because the cMyc promoter has putative binding sites that are available to Zinc finger binding. Sall2, another emerging cancer player in the Sall family, binds to the cMyc promoter region and represses cMyc expression. Thereby, sal/SALL4 may activate JNK signaling through the repression of puc, which is activated by dMyc in Drosophila (Sun, 2020).

    Cell competition occurs when Myc is unevenly distributed between cells. Clones expressing high levels of Myc expand and eliminate the surrounding cells by apoptosis. On the contrary, downregulation of Myc in clones leads to their elimination. Given sal/SALL4-expressing cells are relatively lower Myc expression, it is possible that the surrounding cells with higher Myc expression become competitors and eliminate those lower Myc expression cells. Intriguingly, sal/SALL4-induced migrating cells are not dead and inhibiting cell death cannot repress sal/SALL4-induced cell invasion, so the mechanism may not be apoptosis-driven cell elimination. Previous studies found that JNK activation in surrounding wild-type cells promotes elimination of their neighboring scrib mutants by activating the PVR-ELMO/Mbc-mediated engulfment pathway, and the surrounding JNK is independent of JNK activation in mutant clones. Distinct from this, sal/SALL4-activated non-autonomous activation of JNK is dependent on JNK activation in sal/SALL4-expressing cells. Whether JNK-dependent engulfment plays a major role in sal/SALL4-mediated extrusion needs to be addressed in the future (Sun, 2020).

    Novel function of N-acetyltransferase for microtubule stability and JNK signaling in Drosophila organ development

    Regulation of microtubule stability is crucial for the maintenance of cell structure and function. This study identified an N-terminal acetyltransferase, Mnat9, that regulates cell signaling and microtubule stability in Drosophila. Loss of Mnat9 causes severe developmental defects in multiple tissues. In the wing imaginal disc, Mnat9 RNAi leads to the ectopic activation of c-Jun N-terminal kinase (JNK) signaling and apoptotic cell death. These defects are suppressed by reducing the level of JNK signaling. Overexpression of Mnat9 can also inhibit JNK signaling. Mnat9 colocalizes with mitotic spindles, and its loss results in various spindle defects during mitosis in the syncytial embryo. Furthermore, overexpression of Mnat9 enhances microtubule stability. Mnat9 is physically associated with microtubules and shows a catalytic activity in acetylating N-terminal peptides of α- and β-tubulin in vitro. Cell death and tissue loss in Mnat9-depleted wing discs are restored by reducing the severing protein Spastin, suggesting that Mnat9 protects microtubules from its severing activity. Remarkably, Mnat9 mutated in the acetyl-CoA binding site is as functional as its wild-type form. This study also found that human NAT9 can rescue Mnat9 RNAi phenotypes in flies, indicating their functional conservation. Taken together, it is proposed that Mnat9 is required for microtubule stability and regulation of JNK signaling to promote cell survival in developing Drosophila organs (Mok, 2021).

    ZnT7 RNAi favors Raf(GOF)scrib(-/-)-induced tumor growth and invasion in Drosophila through JNK signaling pathway

    The disruption of zinc homeostasis has been identified in patients suffering from various cancers, but a causative relationship has not yet been established. Drosophila melanogaster has become a powerful model to study cancer biology. Using a Drosophila model of malignant tumor RafGOFscrib-/-, it was observed that the tumor growth, invasion and migration were enhanced by silencing dZnT7, a zinc transporter localized on the Golgi apparatus. Further study indicated that the zinc deficiency in Golgi of dZnT7 RNAi resulted in ER stress which could activate the c-Jun-N-terminal Kinase (JNK) signaling and this process is mediated by Atg9. Lastly, it was demonstrated that the exacerbation of dZnT7 RNAi on tumor was promoted by JNK signaling-dependent cell autonomous and non-autonomous autophagy. These findings suggest that zinc homeostasis in secretory compartments may provide a new therapeutic target for tumor treatment (Wei, 2021).

    Dpp and Hedgehog promote the glial response to neuronal apoptosis in the developing Drosophila visual system

    Damage in the nervous system induces a stereotypical response that is mediated by glial cells. This study used the eye disc of Drosophila melanogaster as a model to explore the mechanisms involved in promoting glial cell response after neuronal cell death induction. These cells rapidly respond to neuronal apoptosis by increasing in number and undergoing morphological changes, which will ultimately grant them phagocytic abilities. This glial response is controlled by the activity of Decapentaplegic (Dpp) and Hedgehog (Hh) signalling pathways. These pathways are activated after cell death induction, and their functions are necessary to induce glial cell proliferation and migration to the eye discs. The latter of these 2 processes depend on the function of the c-Jun N-terminal kinase (JNK) pathway, which is activated by Dpp signalling. Evidence is presented that a similar mechanism controls glial response upon apoptosis induction in the leg discs, suggesting that these results uncover a mechanism that might be involved in controlling glial cells response to neuronal cell death in different regions of the peripheral nervous system (PNS) (Velarde, 2021).

    Derlin-1 and TER94/VCP/p97 are required for intestinal homeostasis

    Adult stem cells are critical for the maintenance of residential tissue homeostasis and functions. However, the roles of cellular protein homeostasis maintenance in stem cell proliferation and tissue homeostasis are not fully understood. This study found that Derlin-1 and TER94/VCP/p97, components of the ER-associated degradation (ERAD) pathway, restrain intestinal stem cell proliferation to maintain intestinal homeostasis in adult Drosophila. Depleting any of them results in increased stem cell proliferation and midgut homeostasis disruption. Derlin-1 is specifically expressed in the ER of progenitors and its C-terminus is required for its function. Interestingly, it was found that increased stem cell proliferation results from elevated ROS levels and activated JNK signaling in Derlin-1- or TER94-deficient progenitors. Further removal of ROS or inhibition of JNK signaling almost completely suppressed increased stem cell proliferation. Together, these data demonstrate that the ERAD pathway is critical for stem cell proliferation and tissue homeostasis. Thus this study provides insights into understanding of the mechanisms underlying cellular protein homeostasis maintenance (ER protein quality control) in tissue homeostasis and tumor development (Liu, 2021).

    Necrosis-induced apoptosis promotes regeneration in Drosophila wing imaginal discs

    Regeneration is a complex process that requires a coordinated genetic response to tissue loss. Signals from dying cells are crucial to this process and are best understood in the context of regeneration following programmed cell death, like apoptosis. Conversely, regeneration following unregulated forms of death, such as necrosis, have yet to be fully explored. This study has developed a method to investigate regeneration following necrosis using the Drosophila wing imaginal disc. Necrosis is shown to stimulate regeneration at an equivalent level to that of apoptosis-mediated cell death and activates a similar response at the wound edge involving localized JNK signaling. Unexpectedly, however, necrosis also results in significant apoptosis far from the site of ablation, which this study terms necrosis-induced apoptosis (NiA). This apoptosis occurs independent of changes at the wound edge and importantly does not rely on JNK signaling. Furthermore, it was found that blocking NiA limits proliferation and subsequently inhibits regeneration, suggesting that tissues damaged by necrosis can activate programmed cell death at a distance from the injury to promote regeneration (Klemm, 2021).

    Anchor maintains gut homeostasis by restricting the JNK and Notch pathways in Drosophila

    The adult Drosophila intestinal epithelium must be tightly regulated to maintain regeneration and homeostasis. The dysregulation of the regenerative capacity is frequently associated with intestinal diseases such as inflammation and tumorigenesis. This study shows that the G protein-coupled receptor Anchor maintains Drosophila adult midgut homeostasis by restricting Jun-N-terminal kinase (JNK) and Notch pathway activity. anchor inactivation resulted in aberrant JNK pathway activation, which led to excessive enteroblast (EB) production and premature enterocyte (EC) differentiation. In addition, increased Notch levels promoted premature EC differentiation following the loss of anchor. This defect induced by the loss of anchor ultimately caused sensitivity to stress or environmental challenge in adult flies. Taken together, these results demonstrate that the activity of anchor is essential to coordinate stem cell differentiation and proliferation to maintain intestinal homeostasis (Wang, 2021).

    Distinct roles of Bendless in regulating FSC niche competition and daughter cell differentiation

    A major goal in the study of adult stem cells is to understand how cell fates are specified at the proper time and place to facilitate tissue homeostasis. This study found that an E2 ubiquitin ligase, Bendless (Ben), has multiple roles in the Drosophila ovarian epithelial follicle stem cell (FSC) lineage. First, Ben is part of the JNK signaling pathway, and it, as well as other JNK pathway genes, were found to be essential for differentiation of FSC daughter cells. The data suggest that JNK signaling promotes differentiation by suppressing the activation of the EGFR effector, ERK. Loss of ben, but not the JNK kinase hemipterous, resulted in an upregulation of hedgehog signaling, increased proliferation and increased niche competition. Lastly, it was demonstrate that the hypercompetition phenotype caused by loss of ben is suppressed by decreasing the rate of proliferation or knockdown of the hedgehog pathway effector, Smoothened (Smo). Taken together, these findings reveal a new layer of regulation in which a single gene influences cell signaling at multiple stages of differentiation in the early FSC lineage (Tatapudy, 2021).

    Dissection of the Regulatory Elements of the Complex Expression Pattern of Puckered, a Dual-Specificity JNK Phosphatase

    For developmental processes, most of the gene networks controlling specific cell responses. It still has to be determined how these networks cooperate and how signals become integrated. The JNK pathway is one of the key elements modulating cellular responses during development. Yet, still little is known about how the core components of the pathway interact with additional regulators or how this network modulates cellular responses in the whole organism in homeostasis or during tissue morphogenesis. A promoter analysis was performed, searching for potential regulatory sequences of puckered (puc) and identified different specific enhancers directing gene expression in different tissues and at different developmental times. Remarkably, some of these domains respond to the JNK activity, but not all. Altogether, these analyses show that puc expression regulation is very complex and that JNK activities participate in non-previously known processes during the development of Drosophila (Karkali, 2021).


    Ahmed-de-Prado, S., Diaz-Garcia, S. and Baonza, A. (2018). JNK and JAK/STAT signalling are required for inducing loss of cell fate specification during imaginal wing discs regeneration in Drosophila melanogaster. Dev Biol 441(1):31-41. PubMed ID: 29870691

    Ai, X., Wang, D., Zhang, J. and Shen, J. (2020). Hippo signaling promotes Ets21c-dependent apical cell extrusion in the Drosophila wing disc. Development 147(22). PubMed ID: 33028612

    Akiyama, T. and Gibson, M. C. (2015). Decapentaplegic and growth control in the developing Drosophila wing. Nature 527(7578):375-8. PubMed ID: 26550824

    Bakal, C., et al. (2008). Phosphorylation networks regulating JNK activity in diverse genetic backgrounds. Science 322(5900): 453-6. PubMed Citation: 18927396

    Bodenmiller, B., et al. (2007). An integrated chemical, mass spectrometric and computational strategy for (quantitative) phosphoproteomics: application to Drosophila melanogaster Kc167 cells. Mol. Biosyst. 3(4): 275-86. PubMed Citation: 17372656

    Feng, Y., Li, Z., Lv, L., Du, A., Lin, Z., Ye, X., Lin, Y. and Lin, X. (2018). Tankyrase regulates apoptosis by activating JNK signaling in Drosophila. Biochem Biophys Res Commun. PubMed ID: 29953853

    Karkali, K. and Martin-Blanco, E. (2021). Dissection of the Regulatory Elements of the Complex Expression Pattern of Puckered, a Dual-Specificity JNK Phosphatase. Int J Mol Sci 22(22). PubMed ID: 34830088

    Klemm, J., Stinchfield, M. J. and Harris, R. E. (2021). Necrosis-induced apoptosis promotes regeneration in Drosophila wing imaginal discs. Genetics 219(3). PubMed ID: 34740246

    Lee, C. W., Kwon, Y. C., Lee, Y., Park, M. Y. and Choe, K. M. (2019). cdc37 is essential for JNK pathway activation and wound closure in Drosophila. Mol Biol Cell: mbcE18120822. PubMed ID: 31483695

    Lee, J. H., Lee, C. W., Park, S. H. and Choe, K. M. (2017). Spatiotemporal regulation of cell fusion by JNK and JAK/STAT signaling during Drosophila wound healing. J Cell Sci [Epub ahead of print]. PubMed ID: 28424232

    Liao, P. C., Tandarich, L. C. and Hollenbeck, P. J. (2017). ROS regulation of axonal mitochondrial transport is mediated by Ca2+ and JNK in Drosophila. PLoS One 12(5): e0178105. PubMed ID: 28542430

    Liu, F., Zhao, H., Kong, R., Shi, L., Li, Z., Ma, R., Zhao, H. and Li, Z. (2021). Derlin-1 and TER94/VCP/p97 are required for intestinal homeostasis. J Genet Genomics. PubMed ID: 34547438.

    Mok, J. W. and Choi, K. W. (2021). Novel function of N-acetyltransferase for microtubule stability and JNK signaling in Drosophila organ development. Proc Natl Acad Sci U S A 118(4). PubMed ID: 33479178

    Muzzopappa, M., Murcia, L. and Milan, M. (2017). Feedback amplification loop drives malignant growth in epithelial tissues. Proc Natl Acad Sci U S A. PubMed ID: 28808034

    Ren, P., Li, W. and Xue, L. (2017). GLYAT regulates JNK-mediated cell death in Drosophila. Sci Rep 7(1): 5183. PubMed ID: 28701716

    Rousset, R., Carballes, F., Parassol, N., Schaub, S., Cerezo, D. and Noselli, S. (2017). Signalling crosstalk at the leading edge controls tissue closure dynamics in the Drosophila embryo. PLoS Genet 13(2): e1006640. PubMed ID: 28231245

    Singh, S.R., Zeng, X., Zhao, J., Liu, Y., Hou, G., Liu, H. and Hou, S.X. (2016). The lipolysis pathway sustains normal and transformed stem cells in adult Drosophila. Nature 538(7623):109-113. PubMed ID: 27680705

    Sun, J., Zhang, J., Wang, D. and Shen, J. (2020). The transcription factor Spalt and human homologue SALL4 induce cell invasion via the dMyc-JNK pathway in Drosophila. Biol Open. PubMed ID: 32098783

    Tatapudy, S., Peralta, J. and Nystul, T. (2021). Distinct roles of Bendless in regulating FSC niche competition and daughter cell differentiation. Development 148(22). PubMed ID: 35020878

    Tavares, L., Correia, A., Santos, M. A., Relvas, J. B. and Pereira, P. S. (2017). dMyc is required in retinal progenitors to prevent JNK-mediated retinal glial activation. PLoS Genet 13(3): e1006647. PubMed ID: 28267791

    Velarde, S. B., Quevedo, A., Estella, C. and Baonza, A. (2021). Dpp and Hedgehog promote the glial response to neuronal apoptosis in the developing Drosophila visual system. PLoS Biol 19(8): e3001367. PubMed ID: 34379617

    Wang, J., Liu, Q., Gong, Y. and Jin, L. H. (2021). Anchor maintains gut homeostasis by restricting the JNK and Notch pathways in Drosophila. J Insect Physiol 134: 104309. PubMed ID: 34496279

    Wang, X., Sun, Y., Han, S., Wu, C., Ma, Y., Zhao, Y., Shao, Y., Chen, Y., Kong, L., Li, W., Zhang, F. and Xue, L. (2017). Amyloid precursor like protein-1 promotes JNK-mediated cell migration in Drosophila. Oncotarget [Epub ahead of print]. PubMed ID: 28537903

    Wei, T., Ji, X., Gao, Y., Zhu, X. and Xiao, G. (2021). ZnT7 RNAi favors Raf(GOF)scrib(-/-)-induced tumor growth and invasion in Drosophila through JNK signaling pathway. Oncogene. PubMed ID: 33649534

    Willsey, H. R., Zheng, X., Carlos Pastor-Pareja, J., Willsey, A. J., Beachy, P. A. and Xu, T. (2016). Localized JNK signaling regulates organ size during development. Elife 5 [Epub ahead of print]. PubMed ID: 26974344

    Yamamoto, M., Ohsawa, S., Kunimasa, K. and Igaki, T. (2017). The ligand Sas and its receptor PTP10D drive tumour-suppressive cell competition. Nature 542(7640): 246-250. PubMed ID: 28092921

    Zygotically transcribed genes

    Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

    The Interactive Fly resides on the
    Society for Developmental Biology's Web server.