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

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

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).

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).


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

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

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

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

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

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

Zygotically transcribed genes

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