JNK pathway

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 (bsk^RNAi#1or2), by expression of JNK phosphatase (puc), or by expression of a dominant negative bsk (bsk^DN). 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 hep^r75/Y animals, while pupal lethal, also have smaller wing discs, as do animals with reduced JNK signaling due to bsk^DN expression. Importantly, total body size is not affected by inhibiting JNK in the wing. Even for the smallest wings generated (rn-Gal4, UAS-bsk^DN), 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 jun^RNAi 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>Ci^ACT 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 (esg^ts > 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 (Raf^gof) also accelerated apoptotic cell death of hyperplastic ISCs (Singh, 2016).

Expressing a constitutively active form of Ras oncogene at 85D (also known as Ras^V12) 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 Ras^V12-transformed RNSCs or in ISCs expressing a dominant-negative form of Notch (N^DN) 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 (esg^ts wg^ts > X). Knockdown of these genes using RNA interference (RNAi) in stem cells ablated most of the stem cells in 1 week. However, expressing Arf79F^RNAi 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 Arf79F^RNAi in Ras^V12-transformed RNSCs ablated almost all of the transformed stem cells. Similarly, expressing Arf79F^RNAi in N^DN-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 Acsl^RNAi in Ras^V12-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-COP^RNAi 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 (hop^Tum-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 esg^ts wg^ts > Acsl^RNAi 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 esg^ts > N^DN + Arf79F^RNAi but not esg^ts flies, indicating that the transformed stem cells were dying through necrosis (Singh, 2016)

Similarly, DHE signals were detected only in ISCs from esg^ts > Arf79F^RNAi 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 Ras^V12-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 (Ras^V12-RNSCs, N^DN-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 Ras^V12-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 yki^B5 mutant clones grow poorly in the wing and eye discs. Consistent with these reports, the current results showed small yki^RNAi and yki^B5 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 yki^RNAi 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).

Ets21C sustains a pro-regenerative transcriptional program in blastema cells of Drosophila imaginal discs

An important unanswered question in regenerative biology is to what extent regeneration is accomplished by the reactivation of gene regulatory networks used during development versus the activation of regeneration-specific transcriptional programs. Following damage, Drosophila imaginal discs, the larval precursors of adult structures, can regenerate missing portions by localized proliferation of damage-adjacent tissue. Using single-cell transcriptomics in regenerating wing discs, a comprehensive view of the transcriptome of regenerating discs was obtained and two regeneration-specific cell populations within the blastema, Blastema1 and Blastema2 were identified. Collectively, these cells upregulate multiple genes encoding secreted proteins that promote regeneration including Pvf1, upd3, asperous, Mmp1, and the maturation delaying factor Ilp8. Expression of the transcription factor Ets21C is restricted to this regenerative secretory zone; it is not expressed in undamaged discs. Ets21C expression is activated by the JNK/AP-1 pathway, and it can function in a type 1 coherent feedforward loop with AP-1 to sustain expression of downstream genes. Without Ets21C function, the blastema cells fail to maintain the expression of a number of genes, which leads to premature differentiation and severely compromised regeneration. As Ets21C is dispensable for normal development, these observations indicate that Ets21C orchestrates a regeneration-specific gene regulatory network. This study has also identified cells resembling both Blastema1 and Blastema2 in scribble tumorous discs. They express the Ets21C-dependent gene regulatory network, and eliminating Ets21C function reduces tumorous growth. Thus, mechanisms that function during regeneration can be co-opted by tumors to promote aberrant growth (Worley, 2022).

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 Raf^GOFscrib^-/-, 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).

Integrins Cooperate With the EGFR/Ras Pathway to Preserve Epithelia Survival and Architecture in Development and Oncogenesis

Adhesion to the extracellular matrix (ECM) is required for normal epithelial cell survival. Disruption of this interaction leads to a specific type of apoptosis known as anoikis. Yet, there are physiological and pathological situations in which cells not connected to the ECM are protected from anoikis, such as during cell migration or metastasis. The main receptors transmitting signals from the ECM are members of the integrin family. However, although integrin-mediated cell-ECM anchorage has been long recognized as crucial for epithelial cell survival, the in vivo significance of this interaction remains to be weighed. This study used the Drosophila wing imaginal disc epithelium to analyze the importance of integrins as survival factors during epithelia morphogenesis. Reducing integrin expression in the wing disc induces caspase-dependent cell death and basal extrusion of the dead cells. In this case, anoikis is mediated by the activation of the JNK pathway, which in turn triggers expression of the proapoptotic protein Hid. In addition, the results strongly suggest that, during wing disc morphogenesis, the EGFR pathway protects cells undergoing cell shape changes upon ECM detachment from anoikis. Furthermore, it was shown that oncogenic activation of the EGFR/Ras pathway in integrin mutant cells rescues them from apoptosis while promoting their extrusion from the epithelium. Altogether, these results support the idea that integrins promote cell survival during normal tissue morphogenesis and prevent the extrusion of transformed cells (Valencia-Exposito, 2022).

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).

Actin remodeling mediates ROS production and JNK activation to drive apoptosis-induced proliferation

Stress-induced cell death, mainly apoptosis, and its subsequent tissue repair is interlinked although knowledge of this connection is still very limited. An intriguing finding is apoptosis-induced proliferation (AiP), an evolutionary conserved mechanism employed by apoptotic cells to trigger compensatory proliferation of their neighboring cells. Studies using Drosophila as a model organism have revealed that apoptotic caspases and c-Jun N-terminal kinase (JNK) signaling play critical roles to activate AiP. For example, the initiator caspase Dronc, the caspase-9 ortholog in Drosophila, promotes activation of JNK leading to release of mitogenic signals and AiP. Recent studies further revealed that Dronc relocates to the cell cortex via Myo1D, an unconventional myosin, and stimulates production of reactive oxygen species (ROS) to trigger AiP. During this process, ROS can attract hemocytes, the Drosophila macrophages, which further amplify JNK signaling cell non-autonomously. However, the intrinsic components connecting Dronc, ROS and JNK within the stressed signal-producing cells remain elusive. This study identified LIM domain kinase 1 (LIMK1), a kinase promoting cellular F-actin polymerization, as a novel regulator of AiP. F-actin accumulates in a Dronc-dependent manner in response to apoptotic stress. Suppression of F-actin polymerization in stressed cells by knocking down LIMK1 or expressing Cofilin, an inhibitor of F-actin elongation, blocks ROS production and JNK activation, hence AiP. Furthermore, Dronc and LIMK1 genetically interact. Co-expression of Dronc and LIMK1 drives F-actin accumulation, ROS production and JNK activation. Interestingly, these synergistic effects between Dronc and LIMK1 depend on Myo1D. Therefore, F-actin remodeling plays an important role mediating caspase-driven ROS production and JNK activation in the process of AiP (Farrell, 2022).

Bilateral JNK activation is a hallmark of interface surveillance and promotes elimination of aberrant cells

Tissue-intrinsic defense mechanisms eliminate aberrant cells from epithelia and thereby maintain the health of developing tissues or adult organisms. 'Interface surveillance' comprises one such distinct mechanism that specifically guards against aberrant cells which undergo inappropriate cell fate and differentiation programs. The cellular mechanisms which facilitate detection and elimination of these aberrant cells are currently unknown. This study findd that in Drosophila imaginal discs, clones of cells with inappropriate activation of cell fate programs induce bilateral JNK activation at clonal interfaces, where wild type and aberrant cells make contact. JNK activation is required to drive apoptotic elimination of interface cells. Importantly, JNK activity and apoptosis are highest in interface cells within small aberrant clones, which likely supports the successful elimination of aberrant cells when they arise. These findings are consistent with a model where clone size affects the topology of interface contacts and thereby the strength of JNK activation in wild type and aberrant interface cells. Bilateral JNK activation is unique to 'interface surveillance' and is not observed in other tissue-intrinsic defense mechanisms, such as classical 'cell-cell competition'. Thus, bilateral JNK interface signaling provides an independent tissue-level mechanism to eliminate cells with inappropriate developmental fate but normal cellular fitness. Finally, oncogenic Ras-expressing clones activate 'interface surveillance' but evade elimination by bilateral JNK activation. Combined, this work establishes bilateral JNK interface signaling and interface apoptosis as a new hallmark of interface surveillance and highlights how oncogenic mutations evade tumor suppressor function encoded by this tissue-intrinsic surveillance system (Prasad, 2023).

Slik maintains tissue homeostasis by preventing JNK-mediated apoptosis

The c-Jun N-terminal kinase (JNK) pathway is an evolutionarily conserved regulator of cell death, which is essential for coordinating tissue homeostasis. This study characterized the Drosophila Ste20-like kinase Slik as a novel modulator of JNK pathway-mediated apoptotic cell death. First, ectopic JNK signaling-triggered cell death is enhanced by slik depletion whereas suppressed by Slik overexpression. Second, loss of slik activates JNK signaling, which results in enhanced apoptosis and impaired tissue homeostasis. In addition, genetic epistasis analysis suggests that Slik acts upstream of or in parallel to Hep to regulate JNK-mediated apoptotic cell death. Moreover, Slik is necessary and sufficient for preventing physiologic JNK signaling-mediated cell death in development. Furthermore, introduction of STK10, the human ortholog of Slik, into Drosophila restores slik depletion-induced cell death and compromised tissue homeostasis. Lastly, knockdown of STK10 in human cancer cells also leads to JNK activation, which is cancelled by expression of Slik. This study has uncovered an evolutionarily conserved role of Slik/STK10 in blocking JNK signaling, which is required for cell death inhibition and tissue homeostasis maintenance in development (Li, 2023).

Lack of apoptosis leads to cellular senescence and tumorigenesis in Drosophila epithelial cells

Programmed cell death (apoptosis) is a homeostasis program of animal tissues designed to remove cells that are unwanted or are damaged by physiological insults. To assess the functional role of apoptosis, the consequences were studied of subjecting Drosophila epithelial cells defective in apoptosis to stress or genetic perturbations that normally cause massive cell death. Many of those cells acquire persistent activity of the JNK pathway, which drives them into senescent status, characterized by arrest of cell division, cell hypertrophy, Senescent Associated β-gal activity (SA-β-gal), reactive oxygen species (ROS) production, Senescent Associated Secretory Phenotype (SASP) and migratory behaviour. Two classes of senescent cells were identified in the wing disc: 1) those that localize to the appendage part of the disc, express the upd, wg and dpp signalling genes and generate tumour overgrowths, and 2) those located in the thoracic region do not express wg and dpp nor they induce tumour overgrowths. Whether to become tumorigenic or non-tumorigenic depends on the original identity of the cell prior to the transformation. The p53 gene was also found to contribute to senescence by enhancing the activity of JNK (Garcia-Arias, 2023).

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).

Senescent cells and macrophages cooperate through a multi-kinase signaling network to promote intestinal transformation in Drosophila

Cellular senescence is a conserved biological process essential for embryonic development, tissue remodeling, repair, and a key regulator of aging. Senescence also plays a crucial role in cancer, though this role can be tumor-suppressive or tumor-promoting, depending on the genetic context and the microenvironment. The highly heterogeneous, dynamic, and context-dependent nature of senescence-associated features and the relatively small numbers of senescent cells in tissues makes in vivo mechanistic studies of senescence challenging. As a result, which senescence-associated features are observed in which disease contexts and how they contribute to disease phenotypes remain largely unknown. Similarly, the specific mechanisms by which various senescence-inducing signals are integrated in vivo to induce senescence and why some cells become senescent while their immediate neighbors do not are unclear. This study identified a small number of cells that exhibit multiple features of senescence in a genetically complex model of intestinal transformation established in the developing Drosophila larval hindgut epithelium. These cells emerge in response to concurrent activation of AKT, JNK, and DNA damage response pathways within transformed tissue. Eliminating senescent cells, genetically or by treatment with senolytic compounds, reduces overgrowth and improves survival. This tumor-promoting role is mediated by Drosophila macrophages recruited to the transformed tissue by senescent cells, which results in non-autonomous activation of JNK signaling within the transformed epithelium. These findings emphasize complex cell-cell interactions underlying epithelial transformation and identify senescent cell-macrophage interactions as a potential druggable node in cancer. It is concluded that interactions between transformed senescent cells and macrophages drive tumorigenesis (Datta, 2023).

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).

The segregation of FSC and daughter cell fates requires the orchestration of multiple developmental signaling pathways to ensure that cell fate specification and proliferation occurs at the correct time and place. This study has identified a new layer of regulation in which a single gene, ben, has multiple distinct roles in promoting differentiation and proliferation in the early FSC lineage (Tatapudy, 2021).

First, the findings demonstrate that ben functions along with the rest of the JNK signaling pathway to promote pFC (posterior follicle cell) differentiation. The phenotypes observed in JNK pathway mutants strongly suggest that the primary defect is a failure to differentiate into mature stalk cells. The range of tube-like and expanded stalk morphological phenotypes that were observed could both be explained by an inability of cells in the stalk regions between follicles to facilitate the pinching off of follicles from the germarium or to intercalate into a single row after the pinching off process has been initiated. The possibility cannot be ruled out that differentiation toward the polar cell fate or main body cell fate are also impaired upon loss of JNK signaling, but no evidence was seen for this with the markers used. Notably, although RNAi knockdown of ben or hep impaired the activity of a reporter for Notch signaling, NRE-GFP, in Region 2b pFCs and mature polar cells, this did not appear to interfere with the ability of mutant pFCs to differentiate into polar cells, so the significance of this effect on cell fate specification is unclear. Additional reporters of Notch signaling may help to determine the extent to which Notch signaling is disrupted in these mutant conditions. It was also found that pERK is retained in pFCs of ovarioles with impaired JNK signaling, and that constitutive activation of pERK phenocopies the differentiation defects observed in JNK pathway mutants. These observations suggest that JNK signaling promotes pFC differentiation into stalk cells by inhibiting the activation of ERK in pFCs (Tat in a JNK-independent manner to help shape the gradient of Hh signaling in FSCs and pFCs. Indeed, it was found that levels of Ptc-pelican-GFP, indicative of Hh signaling, are higher in ben mutant FSCs compared with wild type, and the Ptc-pelican-GFP reporter remains detectable throughout Regions 2b and 3 of tapudy, 2021).

Second, ben functions in the germarium. Likewise, it was found that RNAi knockdown of ben caused the main body follicle cells to retain zfh1 expression, a downstream target of Hh signaling, throughout the germarium and even into Stage 2 in some cases, where it is completely absent in wild-type tissue. However, it was also found that ben mutant main body follicle cells were able to mature into a Cas+, Eya- state, indicating proper differentiation with respect to these two markers. Thus, although loss of ben does not fully impair main body follicle cell differentiation, it may influence the process through its role as an upstream regulator of zfh1 expression. Further study will be required to understand the functional significance of decreased cas and zfh1 expression during main body follicle cell differentiation (Tatapudy, 2021).

JNK signaling has been found to be involved in a wide variety of biological processes in a cell type- and context-dependent manner. Some of the most well-known functions of JNK signaling involve responses to non-homeostatic conditions, such as stress, cellular damage, infection and tumor growth. However, JNK signaling has also been found to be important for processes that occur during normal development or adult homeostasis. Indeed, hep mutants were originally described for their role in dorsal closure of the epidermis in the embryo, and subsequent studies have clearly established the importance of the JNK signaling pathway in this process. Likewise, JNK signaling is also important during adult homeostasis in mammalian stem cell lineages, for example to promote self-renewal of human hematopoietic stem cells and differentiation in the mouse intestinal stem cell and human neural stem cell lineages. In line with this type of role for JNK signaling, the current findings reveal new pathway interactions for JNK signaling in the regulation of cell fate decisions in an epithelial stem cell lineage (Tatapudy, 2021).

In addition to regulating cell fate decisions, a JNK-independent role was identified for Ben in the regulation of proliferation rate and FSC niche competition. Previous studies have established that increased proliferation causes hypercompetition, and that the proliferative response to Hh signaling is the key mediator of the FSC niche competition phenotypes in Hh pathway mutants. Therefore, the hypercompetition phenotype of ben mutant clones is likely caused, in large part, by the increased Hh signaling levels and proliferation rates in these mutants. The findings that overexpression of dap or RNAi knockdown of smo was sufficient to suppress the hypercompetition phenotype of benA mutant clones are consistent with this. It is possible that delayed differentiation in newly-produced ben mutant pFCs also contributes to the hypercompetition phenotype. However, the observation that hep^G0107 mutant clones are not hypercompetitive indicates that the hypercompetition phenotype of ben mutant clones is not caused by a block in the JNK-dependent functions of Ben in pFC differentiation (Tatapudy, 2021).

Taken together, these findings indicate that Ben has JNK-dependent and -independent roles in regulating pFC cell fate decisions as they differentiate into polar, stalk and main body follicle cells. Specifically, the findings support a model in which JNK signaling promotes the differentiation of pFCs during normal homeostasis by helping to downregulate EGFR signaling in early pFCs. Previous studies found that pERK inhibits Groucho and that Groucho is required for the upregulation of Notch signaling in pFCs. Thus, the retention of pERK in pFCs may explain why Notch signaling is not upregulated in JNK pathway mutants. Separately, Ben inhibits Hh signaling in a JNK-independent manner, thereby controlling pFC proliferation, regulating FSC niche competition, and possibly contributing to main body follicle cell differentiation. Thus, this study has revealed a new layer of regulation within the signaling network that controls cell behavior in the early FSC lineage. The recently published single-cell atlases of the adult Drosophila ovary revealed a continuum of changes in gene expression that occur during cellular differentiation in the early FSC lineage, and provide the tools for characterizing this signaling network in a cell-type specific manner. In future studies, it will be interesting to apply these new single-cell approaches to mutants such as those described in this study to further elucidate the molecular basis of cell fate decisions in the FSC lineage (Tatapudy, 2021).

Gbb Regulates Blood Cell Proliferation and Differentiation through JNK and EGFR Signaling Pathways in the Drosophila Lymph Gland

The Drosophila lymph gland is an ideal model for studying hematopoiesis, and unraveling the mechanisms of Drosophila hematopoiesis can improve understanding of the pathogenesis of human hematopoietic malignancies. Bone morphogenetic protein (BMP) signaling is involved in a variety of biological processes and is highly conserved between Drosophila and mammals. Decapentaplegic (Dpp)/BMP signaling is known to limit posterior signaling center (PSC) cell proliferation by repressing the protooncogene dmyc. However, the role of two other TGF-β family ligands, Glass bottom boat (Gbb) and Screw (Scw), in Drosophila hematopoiesis is currently largely unknown. This study showed that the loss of Gbb in the cortical zone (CZ) induced lamellocyte differentiation by overactivation of the EGFR and JNK pathways and caused excessive differentiation of plasmatocytes, mainly by the hyperactivation of EGFR. Furthermore, it was found that Gbb was also required for preventing the hyperproliferation of the lymph glands by inhibiting the overactivation of the Epidermal Growth Factor Receptor (EGFR) and c-Jun N-terminal Kinase (JNK) pathways. These results further advance understanding of the roles of Gbb protein and the BMP signaling in Drosophila hematopoiesis and the regulatory relationship between the BMP, EGFR, and JNK pathways in the proliferation and differentiation of lymph gland hemocytes (Zhang, 2023).

Mitochondrial metabolism in Drosophila macrophage-like cells regulates body growth via modulation of cytokine and insulin signaling

Macrophages play critical roles in regulating and maintaining tissue and whole-body metabolism in normal and disease states. While the cell-cell signaling pathways that underlie these functions are becoming clear, less is known about how alterations in macrophage metabolism influence their roles as regulators of systemic physiology. This was investigated by examining Drosophila macrophage-like cells called hemocytes. Knockdown of TFAM, a mitochondrial genome transcription factor, to reduce mitochondrial OxPhos activity specifically in larval hemocytes. This reduction in hemocyte OxPhos leads to a decrease in larval growth and body size. These effects are associated with a suppression of systemic insulin, the main endocrine stimulator of body growth. It was also found that TFAM knockdown leads to decreased hemocyte JNK signaling and decreased expression of the TNF alpha homolog, Eiger in hemocytes. Furthermore, genetic knockdown of hemocyte JNK signaling or Eiger expression mimics the effects of TFAM knockdown and leads to a non-autonomous suppression of body size without altering hemocyte numbers. These data suggest that modulation of hemocyte mitochondrial metabolism can determine their non-autonomous effects on organismal growth by altering cytokine and systemic insulin signaling. Given that nutrient availability can control mitochondrial metabolism, these findings may explain how macrophages function as nutrient-responsive regulators of tissue and whole-body physiology and homeostasis (Sriskanthadevan-Pirahas, 2023).

JNK signaling in pioneer neurons organizes ventral nerve cord architecture in Drosophila embryos

Morphogenesis of the Central Nervous System (CNS) is a complex process that obeys precise architectural rules. Yet, the mechanisms dictating these rules remain unknown. Analyzing morphogenesis of the Drosophila embryo Ventral Nerve Cord (VNC), this study observe that a tight control of JNK signaling is essential for attaining the final VNC architecture. JNK signaling in a specific subset of pioneer neurons autonomously regulates the expression of Fasciclin 2 (Fas 2) and Neurexin IV (Nrx IV) adhesion molecules, probably via the transcription factor zfh1. Interfering at any step in this cascade affects fasciculation along pioneer axons, leading to secondary cumulative scaffolding defects during the structural organization of the axonal network. The global disorder of architectural landmarks ultimately influences nervous system condensation. In summary, these data point to JNK signaling in a subset of pioneer neurons as a key element underpinning VNC architecture, revealing critical milestones on the mechanism of control of its structural organization (Karkali, 2023).

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).

Intestine-derived α-synuclein initiates and aggravates pathogenesis of Parkinson's disease in Drosophila

Aberrant aggregation of α-synuclein (α-syn) is a key pathological feature of Parkinson's disease (PD), but the precise role of intestinal α-syn in the progression of PD is unclear. In a number of genetic Drosophila models of PD, α-syn was frequently ectopically expressed in the neural system to investigate the pathobiology. This study investigated the potential role of intestinal α-syn in PD pathogenesis using a Drosophila model. Human α-syn was overexpressed in Drosophila guts, and life span, survival, immunofluorescence and climbing were evaluated. Immunofluorescence, Western blotting and reactive oxygen species (ROS) staining were performed to assess the effects of intestinal α-syn on intestinal dysplasia. High-throughput RNA and 16S rRNA gene sequencing, quantitative RT-PCR, immunofluorescence, and ROS staining were performed to determine the underlying molecular mechanism. It was found that the midgut α-syn alone recapitulated many phenotypic and pathological features of PD, including impaired life span, loss of dopaminergic neurons, and progressive motor defects. The intestine-derived α-syn disrupted intestinal homeostasis and accelerated the onset of intestinal ageing. Moreover, intestinal expression of α-syn induced dysbiosis, while microbiome depletion was efficient to restore intestinal homeostasis and ameliorate the progression of PD. Intestinal α-syn triggered ROS, and eventually led to the activation of the dual oxidase (DUOX)-ROS-Jun N-terminal Kinase (JNK) pathway. In addition, α-syn from both the gut and the brain synergized to accelerate the progression of PD. The intestinal expression of α-syn recapitulates many phenotypic and pathologic features of PD, and induces dysbiosis that aggravates the pathology through the DUOX-ROS-JNK pathway in Drosophila. These findings provide new insights into the role of intestinal α-syn in PD pathophysiology (Liu, 2022).

Novel studies on Drosophila melanogaster model reveal the roles of JNK-Jak/STAT axis and intestinal microbiota in insulin resistance

The JNK pathway play a critical role in insulin resistance induced by a long-term high-sugar diet. However, the roles of up- and downstream molecules of the JNK pathway in insulin resistance are less known in vertebrates and invertebrates. As a classical organism in biological research, Drosophila melanogaster has been widely applied to the studies of mechanism of insulin resistance. Based on previous studies, this study found a novel predictive mechanism of the formation of insulin resistance in D. melanogaster. JNK activated by high-sugar diet and dysregulated intestinal microbiota could mediate inflammation, and then the activated JNK released Upd3, which in turn stimulated Jak/STAT pathway to release ImpL2. ImpL2 can compete with Drosophila insulin-like peptides (Dilps) for binding with the insulin receptor and inhibit the activation of insulin pathway. This study reviewed novel studies on the insulin signalling pathway based on the D. melanogaster model. The findings support the hypothesis. Therefore how a long-term high-sugar diet disrupts intestinal microbiota to induce inflammation and the disruption of JNK-Jak/STAT axis. This description may offer some new clues to the formation of insulin resistance (Meng, 2022).

Hsp70 overexpression in Drosophila hemocytes attenuates benzene-induced immune and developmental toxicity via regulating ROS/JNK signaling pathway

Benzene, a ubiquitous environmental chemical, is known to cause immune dysfunction and developmental defects. This study aims to investigate the relation between benzene-induced immune dysfunction and developmental toxicity in a genetically tractable animal model, Drosophila melanogaster. Further, the study explored the protective role of Heat Shock Protein 70 (Hsp70) against benzene-induced immunotoxicity and subsequent developmental impact. Drosophila larvae exposed to benzene (1.0, 10.0, and 100.0 mM) were examined for total hemocyte (immune cells) count, phagocytic activity, oxidative stress, apoptosis, and their developmental delay and reduction were analyzed. Benzene exposure for 48 h reduced the total hemocytes count and phagocytic activity, along with an increase in the Reactive Oxygen Species (ROS), and lipid peroxidation in the larval hemocytes. Subsequently, JNK-dependent activation of the apoptosis (Caspase-3 dependent) was also observed. During their development, benzene exposure to Drosophila larvae led to 3 days of delay in development, and ~40% reduced adult emergence. Hsp70-overexpression in hemocytes was found to mitigate benzene-induced oxidative stress and abrogated the JNK-mediated apoptosis in hemocytes, thus restoring total hemocyte count and improving phagocytotic activity. Further, hsp70-overexpression in hemocytes also lessened the benzene-induced developmental delay (rescue of 2.5 days) and improved adult emergence (~20%) emergence, revealing a possible control of immune cells on the organism's development and survival. Overall, this study established that hsp70-overexpression in the Drosophila hemocytes confers protection against benzene-induced immune injury via regulating the ROS/JNK signaling pathway, which helps in the organism's survival and development (D'Souza, 2022).

Extracellular matrix protein N-glycosylation mediates immune self-tolerance in Drosophila melanogaster

In order to respond to infection, hosts must distinguish pathogens from their own tissues. This allows for the precise targeting of immune responses against pathogens and also ensures self-tolerance, the ability of the host to protect self tissues from immune damage. One way to maintain self-tolerance is to evolve a self signal and suppress any immune response directed at tissues that carry this signal. This study characterizes the Drosophila tuSz mutant strain, which mounts an aberrant immune response against its own fat body. This study demonstrates that this autoimmunity is the result of two mutations: 1) a mutation in the Glucosidase 1/GCS1 gene that disrupts N-glycosylation of extracellular matrix proteins covering the fat body, and 2) a mutation in the Drosophila Janus Kinase ortholog that causes precocious activation of hemocytes. Data indicate that N-glycans attached to extracellular matrix proteins serve as a self signal and that activated hemocytes attack tissues lacking this signal. The simplicity of this invertebrate self-recognition system and the ubiquity of its constituent parts suggests it may have functional homologs across animals (Mortimer, 2021).

This work has investigated the Drosophila tuSz¹ mutant strain. tuSz¹ is a temperature-sensitive mutant, and at the restrictive temperature, posterior fat body tissue is melanotically encapsulated by hemocytes in a reaction similar to the antiparasitoid immune response. The tuSz¹ phenotype is caused by two tightly linked mutations: a nonconditional, dominant gain-of-function mutation in hop that leads to ectopic immune activation and a temperature-sensitive, recessive mutation in GCS1 that leads to loss of protein N-glycosylation of the ECM overlaying the posterior fat body. These data lead to a a proposal of a two-step model in which immune activation and the loss of SAMP presentation/recognition are both necessary for the breakdown of self-tolerance. In a naïve wild-type larva, neither condition is met and self-tolerance is maintained. In the case of the tuSz¹ mutant, the posterior fat body lacks appropriate ECM protein N-glycosylation and is targeted by constitutively activated hemocytes for encapsulation. This two-step model is also reflected in the hop^Tum mutant background, in which the simultaneous disruption of N-glycosylation in this immune-activated background results in tissue self-encapsulation similar to the tuSz¹ mutant (Mortimer, 2021).

Models describing the necessity for two independent signals in fly encapsulation responses. (A) Homeostasis is maintained in naïve wild-type larvae. (B) In tuSz¹ mutant larvae, immune cells are inappropriately activated by JAK-STAT pathway activation due to the hop^Sz gain-of-function mutation. The loss of protein N-glycosylation in posterior fat body tissue due to the GCS1^Sz mutation leads to loss of self-tolerance and tissue encapsulation. (C) In the model of self-tolerance described in a previous study, the coupled phenotypes of loss of cell integrity and loss of ECM integrity are sufficient to disrupt self-tolerance. (D) Immune cells are activated following parasitoid wasp infection, presumably due to the wound-mediated activation of JAK-STAT signaling. SAMP-presenting host tissues are protected from encapsulation, and wasp eggs may be targeted for encapsulation because they are missing the ECM N-glycosylation SAMP (Mortimer, 2021).

Interestingly, previous work also documented the necessity of at least two signals for self-encapsulation in Drosophila. In that case, both the loss of the ECM (with its glycosylated proteins) and the disrupted positional integrity of the underlying fat body cells (potentially mimicking a wound) were required for immune cells to become activated and encapsulate the self tissue. A similar loss of ECM and underlying cell integrity was also found in the classical melanotic tumor mutant tu(2)W. This model, in which at least two factors are required for self-encapsulation, may explain why the several classically described self-encapsulation mutants, unlike virtually all other types of visible Drosophila mutants, were never successfully mapped (Mortimer, 2021).

The disruption of either factor in the two-step model in isolation is not sufficient to cause self-encapsulation. This can be seen in parasitoid wasp infected larvae; the wounding associated with parasitoid infection leads to immune activation, but in the absence of SAMP disruption, the fly is able to specifically encapsulate the parasitoid egg while protecting against self-encapsulation. Conversely, while internal tissue damage in a naive larva does attract hemocyte interactions, in the absence of an immune stimulus, this does not lead to self-encapsulation, but rather the hemocytes attempt to repair the damaged tissue. That blood cells err on the side of fixing disrupted self tissue rather than treating it as pathogenic and encapsulating it unless another stress signal is also present suggests that flies may have evolved a multi-input system to safeguard against spurious encapsulation (Mortimer, 2021).

D. melanogaster immune responses have proven to be an excellent model for understanding the mechanisms underlying conserved innate immune responses, including those of humans. Findings on Drosophila self-tolerance may also be relevant to human innate self-tolerance. Indeed, data from a range of studies are consistent with the idea that protein glycosylation is a mediator of vertebrate immune responses, and cell-surface glycans have been proposed as candidate SAMPs for the innate immune response to distinguish healthy self tissues from aberrant or foreign tissues even if the mechanisms are not entirely understood. Protein-linked sugar groups should presumably fit this role well, as they can take on diverse combinations of sugar residues and branching patterns (Mortimer, 2021).

Protein N-glycosylation is a complex multistep process that begins with the addition of a presynthesized glycosyl precursor to the protein at an asparagine residue. This nascent glycan is then trimmed back to a core glycan structure, a process that is initiated by the activity of GCS1. The core glycan is then elaborated with the addition of multiple carbohydrate groups to give rise to a variety of final structures, with hybrid and complex type N-glycans among the most prevalent. Glycan elaboration begins with the activity of Mgat1, which leads to the production of hybrid type N-glycans. These hybrid N-glycans can be further processed by the α-mannosidase-I and -II family of enzymes to produce paucimannose N-glycans, which can serve as complex N-glycan precursors and are further elaborated by downstream enzymes to give rise to the final complex N-glycan structure. The current data suggest that disruption of any of the genes encoding key N-glycan-processing enzymes will be associated with the loss of self-tolerance in Drosophila. Similarly, the loss of the α-mannosidase-II (αM-II) gene in mice is linked with the development of an autoimmune phenotype that is likened to systemic lupus erythematosus. Like the tuSz¹ mutant, the αM-II mouse phenotype arises due to alterations in protein N-glycosylation in nonimmune tissues and is mediated by innate immune cells. Altered patterns of protein N-glycosylation are also observed in additional mouse models of autoimmune disease and have been linked to autoimmune disease in human patients (Mortimer, 2021).

The ECM is a conserved structure made up of numerous proteins, many of which are N-glycosylated, including laminin and collagen. A role for the ECM in mediating self-tolerance has been previously proposed: The encapsulation of self tissues in D. melanogaster is also observed following RNA interference (RNAi) knockdown of genes encoding the ECM proteins laminin and collagen, supporting the idea that SAMPs reside in the ECM. The role of the ECM in self-tolerance is further emphasized by tissue transplantation studies in Drosophila. Drosophila larvae are largely tolerant of conspecific tissue transplants, but this tolerance is abolished when tissues are first treated with collagenase to disrupt the ECM, leading to the specific encapsulation of treated tissues. Reactivity to ECM proteins is also associated with various forms of human autoimmune disease. Based on these data, it is proposed that the N-glycosylation of ECM proteins may serve as a conserved self-tolerance signal for innate immune mechanisms and that loss of ECM protein N-glycosylation may lead to loss of self-tolerance and, consequently, autoimmune disease in a diverse range of species (Mortimer, 2021).

An alternative means by which hosts can recognize pathogens is missing-self recognition. Instead of tracking pathogen diversity with numerous recognition receptors, as in nonself recognition, missing-self recognition does not rely on tracking pathogens at all, but only on specifically recognizing self and attacking tissues that lack the self signal. Further, unlike germ line-encoded forms of nonself recognition, missing-self recognition systems allow host species to respond to novel pathogen types that they have never encountered in their evolutionary history. Protein glycosylation plays an important role in the handful of identified missing-self recognition systems of vertebrates. The most well-known case of missing-self recognition involves the interaction between vertebrate NK cells and host MHC class I (MHCI) proteins. All vertebrate cells produce MHCI to display any possible antigens present in their cytoplasm to T cells, but intracellular pathogens often suppress host cell MHCI expression to prevent their molecules from being displayed. NK cells are lymphoid-type cells that induce cytolysis in infected host cells. In an uninfected state, recognition of properly glycosylated MHCI inhibits NK cell cytolysis of host cells, but in an infected state in which host cells are missing the MHCI self signal, the NK cell inhibitory receptors fail to recognize 'self,' and the infected host cells are lysed, an effective means of killing intracellular pathogens that have suppressed host MHC signaling (Mortimer, 2021).

As of yet, there are no examples of missing-self immune recognition systems in invertebrates, and it has been hypothesized that invertebrate immune systems rely largely on PRRs for nonself recognition of pathogens. Still, invertebrates do mount immune responses against a variety of inanimate objects like oil droplets, sterile nylon, and charged chromatography beads as well as tissue transplants from other insect species. All of these foreign bodies presumably lack distinct PAMPs, suggesting that invertebrates have some sort of missing-self recognition system. Additionally, while multiple antimicrobial PRRs have been identified in the Drosophila genome, PRRs targeting macroparasites like parasitoid wasps have not yet been discovered. The current model of self-recognition suggests that following parasitoid infection, activated immune cells assess all exposed tissue surfaces for the self-tolerance glycan signal and that the absence of this Drosophila SAMP on parasitoid wasp eggs might be the cue that targets them for melanotic encapsulation (Mortimer, 2021).

Tumor elimination by clustered microRNAs miR-306 and miR-79 via noncanonical activation of JNK signaling

JNK signaling plays a critical role in both tumor promotion and tumor suppression. This study identified clustered microRNAs (miRNAs) miR-306 and miR-79 as novel tumor-suppressor miRNAs that specifically eliminate JNK-activated tumors in Drosophila. While showing only a slight effect on normal tissue growth, miR-306 and miR-79 strongly suppressed growth of multiple tumor models, including malignant tumors caused by Ras activation and cell polarity defects. Mechanistically, these miRNAs commonly target the mRNA of an E3 ubiquitin ligase ring finger protein 146 (RNF146). RNF146 promotes degradation of tankyrase (Tnks), an ADP-ribose polymerase that promotes JNK activation in a noncanonical manner. Thus, downregulation of RNF146 by miR-306 and miR-79 leads to hyper-enhancement of JNK activation. These data show that, while JNK activity is essential for tumor growth, elevation of miR-306 or miR-79 overactivate JNK signaling to the lethal level via noncanonical JNK pathway and thus eliminate tumors, providing a new miRNA-based strategy against cancer (Wang, 2023).

Distinct signaling signatures drive compensatory proliferation via S-phase acceleration

Regeneration relies on cell proliferation to restore damaged tissues. Multiple signaling pathways activated by local or paracrine cues have been identified to promote regenerative proliferation. How different types of tissue damage may activate distinct signaling pathways and how these differences converge on regenerative proliferation is less well defined. To better understand how tissue damage and proliferative signals are integrated during regeneration, this study investigated models of compensatory proliferation in Drosophila imaginal discs. Compensatory proliferation was found to be associated with a unique cell cycle profile, which is characterized by short G1 and G2 phases and, surprisingly, by acceleration of the S-phase. S-phase acceleration can be induced by two distinct signaling signatures, aligning with inflammatory and non-inflammatory tissue damage. Specifically, non-autonomous activation of JAK/STAT and Myc in response to inflammatory damage, or local activation of Ras/ERK and Hippo/Yki in response to elevated cell death, promote accelerated nucleotide incorporation during S-phase. This previously unappreciated convergence of different damaging insults on the same regenerative cell cycle program reconciles previous conflicting observations on proliferative signaling in different tissue regeneration and tumor models (Crucianelli, 2023).

Drosophila Toll-9 is induced by aging and neurodegeneration to modulate stress signaling and its deficiency exacerbates tau-mediated neurodegeneration

Drosophila Toll-9 is most closely related to mammalian Toll-like receptors; however, physiological functions of Toll-9 remain elusive. This study examined the roles of Toll-9 in fly brains in aging and neurodegeneration. Toll-9 mRNA levels were increased in aged fly heads accompanied by activation of nuclear factor-kappa B (NF-kB) and stress-activated protein kinase (SAPK; Jun-N-terminal Kinase pathway) signaling, and many of these changes were modulated by Toll-9 in glial cells. The loss of Toll-9 did not affect lifespan or brain integrity, whereas it exacerbated hydrogen peroxide-induced lethality. Toll-9 expression was also induced by nerve injury but did not affect acute stress response or glial engulfment activity, suggesting Toll-9 may modulate subsequent neurodegeneration. In a fly tauopathy model, Toll-9 deficiency enhanced neurodegeneration and disease-related tau phosphorylation with reduced SAPK activity, and blocking SAPK enhanced tau phosphorylation and neurodegeneration. In sum, Toll-9 is induced upon aging and nerve injury and affects neurodegeneration by modulating stress kinase signaling (Sakakibara, 2023).

The CRL4 E3 ligase Mahjong/DCAF1 controls cell competition through the transcription factor Xrp1, independently of polarity genes

Cell competition, the elimination of cells surrounded by more fit neighbors, is proposed to suppress tumorigenesis. Mahjong (Mahj), a ubiquitin E3 ligase substrate receptor, has been thought to mediate competition of cells mutated for lethal giant larvae (lgl), a neoplastic tumor suppressor that defines apical-basal polarity of epithelial cells. This study shows that Drosophila cells mutated for mahjong, but not for lgl [l(2)gl], are competed because they express the bZip-domain transcription factor Xrp1, already known to eliminate cells heterozygous for ribosomal protein gene mutations (Rp/+ cells). Xrp1 expression in mahj mutant cells results in activation of JNK signaling, autophagosome accumulation, eIF2α phosphorylation and lower translation, just as in Rp/+ cells. Cells mutated for damage DNA binding-protein 1 (ddb1; pic) or cullin 4 (cul4), which encode E3 ligase partners of Mahj, also display Xrp1-dependent phenotypes, as does knockdown of proteasome subunits. These data suggest a new model of mahj-mediated cell competition that is independent of apical-basal polarity and couples Xrp1 to protein turnover (Kumar, 2022).

Cell competition, which is the elimination of, in most cases, slower-growing cells by faster-growing cells in mosaics, is important for precise development, regeneration and physiological maintenance. Cell competition was first recognized in Drosophila in the case of cells lacking one copy of ribosomal protein genes (Rp/+). These mutants, which are known as 'Minutes' because of their thin body bristles, also display slow growth. Minute mutant cells are eliminated from mosaics with wild-type cells by caspase-dependent cell death. Super-competition, the name given to the process of eliminating wild-type cells, happens in mosaics with faster-growing Myc- or Yorkie (Yki)-expressing cells. Considered together, cell competition and super-competition suggest that comparison of cellular fitness leads to cell competition. Because the mammalian homologs of Myc and Yki are proto-oncogenes, it has been suggested that super-competition might contribute to tumor expansion, as several recent studies have confirmed (Kumar, 2022).

Cell competition may also be tumor suppressive. Global loss of apico-basal polarity genes such as lgl [l(2)gl)] or scribble (scrib) leads to polarity-deficient neoplasia of Drosophila imaginal discs, but clones of lgl or scrib cells are eliminated from mosaics. These lgl or scrib mutant clones do not form tumors unless cell competition is blocked and mutant cells remain in the epithelium (Kumar, 2022).

Competition of Rp/+ mutant cells might also serve a tumor-surveillance role. Rp genes are spread throughout the genome, and it has been shown they can serve as sensors for aneuploidy, leading to elimination of aneuploid cells containing monosomies that affect Rp gene dose. Mutants with disrupted cell competition accumulate aneuploid cells. These would be expected to be tumorigenic in mammals, where aneuploidy is associated with tumorigenesis. Competition of Rp/+ cells depends on the Drosophila bZip AT-hook domain transcription factor Xrp1. The Xrp1 expression induced in Rp/+ imaginal discs is also responsible for most of their altered gene expression, their slow growth and their reduced translation, in addition to their propensity to be eliminated by cell competition. Xrp1 is also expressed in the DNA damage response, where its transcription is p53 dependent. Xrp1 induction in Rp/+ cells is independent of p53 but dependent on a particular Rp protein, RpS12, which is thought to play a role in signaling the defect in ribosome biogenesis (Kumar, 2022).

The tumor-suppressive cell competition of polarity gene mutant cells has been proposed to go through Mahjong (Mahj), a CRL4 E3 ubiquitin ligase (Ly, 2019). mahj physically interacts with Lgl, and its overexpression in lgl mutant clones suppresses their elimination from mosaic tissues. Interestingly, mahj knockdown in MDCK cell cultures also leads to their elimination by co-cultured normal MDCK cells, suggesting a cell competition mechanism that is conserved between Drosophila and mammalian cells (Tamori, 2010). The mammalian homolog of mahj, known as DDB1-Cul4-associated factor 1 (DCAF1) or human immunodeficiency virus type 1 accessory protein Vpr-binding protein (VprBP), is important for G2 cell cycle arrest and virus replication after HIV1 infection. Dcaf1 is required for mouse embryogenesis and its knockdown affects cell proliferation, cell cycle and cell survival in multiple cell types. Dcaf1 interacts with the Hippo pathway and its knockdown also stabilizes p53, but there has been no report of Dcaf1/VprBP affecting epithelial cell polarity in mammals (Kumar, 2022).

In Drosophila, the overall transcriptional signature of mahj mutant wing discs is unexpectedly similar to that of Rp/+ mutants, including upregulation of Xrp1 mRNA. Because mahj and Rp/+ cells were thought to represent distinct mechanisms of cell competition, this finding suggested a gene expression signature common to cells targeted by cell competition. Besides transcription, other similarities have been reported between mahj and Rp/+ mutant cells, including autophagosome accumulation and evidence of proteotoxic stress (Kumar, 2022).

This study shows that mahj mutant cells trigger cell competition through an Xrp1-dependent pathway like that in Rp/+ cells, and distinct from cell competition of lgl or scrib clones, which do not express or depend on Xrp1 function for elimination. Xrp1 expression also makes mahj mutant cells phenotypically like Rp/+ cells, that is, results in 'Minute-like' thin thoracic bristles, slow growth, reduced translation, altered autophagy and increased JNK signaling. Regulation of Xrp1 by mahj likely requires its E3 ligase activity, depending on DNA Damage Binding Protein 1 (Ddb1) and Cullin 4 (Cul4). These results show that mahj mutant cells suffer cell competition because of a transcriptional response to altered ubiquitinylation mediated by Xrp1 and therefore resembling Rp/+ mutant cells. This seems unrelated to elimination of scrib or lgl mutant cells - the polarity-defective cells. Thus, loss of mahj function is an additional genotype triggering elimination by the Xrp1-dependent pathway that also removes Minute cells, not the mechanism for eliminating tumorigenic polarity-deficient cells (Kumar, 2022).

This research explored the cell competition mechanisms of Mahj, a CRL4 ubiquitin ligase (Ly, 2019), the mutation of which triggers similar cellular effects to Rp/+ mutations, including similar changes in gene expression, global translation rates, JNK activity and autophagy, leading mahj cells to be eliminated by competition with wild-type cells, as Rp/+ cells are. The basis of the similarity is that mahj and Rp loss of function both activate expression of Xrp1, the transcription factor that coordinates these effects. Unlike Rp/+ genotypes, which activate Xrp1 through a rpS12-dependent mechanism, mahj regulates Xrp1 most likely through its ubiquitin ligase activity, which depends on DDB1, Cul4 and Roc1a, although the specific ubiquitylated target has not yet been identified. It is suggested that Xrp1 is likely to be activated by a protein, or proteins, that are normally degraded by Mahj-dependent ubiquitylation, because Xrp1 is also activated by inhibition of the proteasome, which is expected to affect the degradation of ubiquitylated proteins, but not other functional consequences of ubiquitylation. The relevant target does not seem to be Warts, despite the fact that levels of Warts and Hippo pathway activity also control cellular growth and global translation levels, and can stimulate cell competition. These studies support the notion that Xrp1 is a sensor of multiple cellular defects that cause cell competition, rather than that of a 'loser signature' common to distinct cell competition mechanisms (Kumar, 2022).

Mahj was previously thought to be responsible for the cell competition of cells mutated for lgl, a gene that controls apical basal cell polarity. mahj was originally linked to apical-basal polarity because of a physical interaction with Lgl, and because mahj overexpression can rescue lgl mutant clones for elimination, suggesting that mahj behaves as an intracellular signal transducer of lgl activity in cell competition.. As such, it was surprising when similar gene expression changes were observed in mahj mutant and Rp mutant wing discs, because these were assumed to reflect distinct cell competition pathways and suggested a common gene expression signature associated with competed cells. This study shows, however, that neither lgl nor scrib, another related cell polarity gene, affects cell competition by the same mechanism as mahj, because neither lgl nor scrib mutant cells express or require Xrp1. Interestingly, several distinct pathways have recently been described to mediate the elimination of scrib mutant cells in competition with wild-type cells, and none of these pathways are shown to be required for the elimination of Rp/+ mutant cells. In addition, mahj loss by itself does not result in apical-basal polarity defects, and its mammalian homolog is implicated in cell cycle regulation, genome integrity and p53 activity. Drosophila mahj, which is an essential gene, regulates neural stem cell reactivation and may have other roles in non-neuronal tissues, as suggested by defects observed when mahj is depleted in posterior wing compartments. Accordingly, it is concluded that mahj mutants affect cellular growth and cell competition in a manner unrelated to lgl and scrib, and that the functional relationship of mahj to apical-basal polarity pathways, should any exist, is unclear. The functional importance of physical interaction between Mahj and Lgl remains to be explored. It is known that lgl clones are rescued by reduced Hippo signaling, although this study did not detect reduced Hippo signaling after mahj overexpression in the absence of lgl mutations (Kumar, 2022).

These studies provide further evidence for Xrp1 as an integrator of multiple seemingly independent cellular defects that each result in a common spectrum of cellular responses and predispose cells to competitive elimination by wild-type neighbors (Kiparaki, 2022). These functional roles for Xrp1 first became apparent through its role in the slow growth, reduced translation and competitive elimination of Rp/+ cells, in which Xrp1 expression is induced in an rpS12-dependent manner. In the case of mahj, Xrp1 protein expression is induced to confer a very similar spectrum of cellular effects, but independently of rpS12 and perhaps depending on stabilization of a protein normally targeted for proteasomal turnover by mahj-dependent ubiquitylation. Xrp1 expression was first found as a p53-regulated gene, perhaps part of the DNA damage response. Recently, Xrp1 induction has also been found as a response to ER stress, possibly through translational regulation downstream of eIF2α phosphorylation. It has been suggested that eIF2α phosphorylation, and Xrp1 expression, can also be triggered by a global, cytoplasmic proteotoxic stress, which is suggested to occur as a consequence of deficient ribosome assembly in Rp mutant cells. Xrp1 expression in response to proteasome inhibition is one piece of evidence for this model. This study shows, however, that Xrp1 is induced, and cell competition results after loss of mahj, a single E3-ligase adapter protein that probably targets only a moderate number of proteins for degradation. Thus, an alternative explanation of Xrp1 induction after proteasome inhibition is that this could reflect stabilization of one or a few specific proteins. Overall, a picture is emerging of Xrp1 as a stress-responsive transcription factor whose expression can be initiated by multiple distinct pathways, then leading to a common cellular response, including the elimination of the stressed cells by competition with nearby wild-type cells, when such cells are available (Kumar, 2022).

Importantly, cells depleted for DCAF1/VprBP, the mammalian homolog of Mahj, are eliminated by competition with wild-type cells in mammalian cell culture. Thus, cell competition of mahj mutant cells may be a conserved process. Conservation of cell competition has not yet been demonstrated for Rp/+ cells in mammals, although it may very well occur. In mammals, knockdown of either mahj or its binding partner ddb1 results in P53 activation, which is functionally required for the resulting phenotypes. Differences in p53 activity lead to cell competition in many mammalian systems. p53 is not required for mahj-mediated cell competition in Drosophila, but because Xrp1 is a target of Drosophila p53 in irradiated cells, it is possible Xrp1 is a p53 target that has replaced the cell competition role of p53 in Drosophila, as has already been suggested for the competition of Rp/+ cells, which is also p53 independent in Drosophila, although Rp mutations activate p53 in mammals. Thus, mahj-mediated cell competition may provide another example where Xrp1 mediates a process in Drosophila that is dependent on p53 in mammals (Kumar, 2022).

MicroRNA miR-274-5p supresses Found-in-Neurons associated with Melanotic mass Formation and Developmental Growth in Drosophila

The hematopoietic system plays a crucial role in immune defense response and normal development, and it is regulated by various factors from other tissues. The dysregulation of hematopoiesis is associated with melanotic mass formation; however, the molecular mechanisms underlying this process are poorly understood. This study observed that the overexpression of miR-274 in the fat body resulted in the formation of melanotic masses. Moreover, abnormal activation of the JNK and JAK/STAT signaling pathways was linked to these consequences. In addition to this defect, miR-274 overexpression in the larval fat body decreased the total tissue size, leading to a reduction in body weight. miR-274-5p was found to directly supress the expression of found-in-neurons (fne), which encodes an RNA-binding protein. Similar to the effects of miR-274 overexpression, fne depletion led to melanotic mass formation and growth reduction. Collectively, miR-274 plays a regulatory role in the fne-JNK signaling axis in melanotic mass formation and growth control (Kim, 2023).

Ectopic expression of matrix metalloproteinases and filopodia extension via JNK activation are involved in the invasion of blood tumor cells in Drosophila mxc mutant

Drosophila mxc(mbn1) mutant exhibits severe hyperplasia in larval hematopoietic tissue called the lymph glands (LGs). However, the malignant nature of these cells remains unknown. This study aimed to identify if mxc(mbn1) LG cells behave as malignant tumor cells and uncover the mechanism(s) underlying the malignancy of the mutant hemocytes. When mutant LG cells were allografted into normal adult abdomens, they continued to proliferate; however, normal LG cells did not proliferate. Mutant circulating hemocytes also attached to the larval central nervous system (CNS), where the basement membrane was disrupted. The mutant hemocytes displayed higher expression of matrix metalloproteinase (MMP) 1 and MMP2 and higher activation of the c-Jun N-terminal kinase (JNK) pathway than normal hemocytes. Depletion of MMPs or JNK mRNAs in LGs resulted in reduced numbers of hemocytes attached to the CNS, suggesting that the invasive phenotype involved elevated expression of MMPs via hyperactivation of the JNK pathway. Moreover, hemocytes with elongated filopodia and extra lamellipodia were frequently observed in the mutant hemolymph, which also depended on JNK signaling. Thus, the MMP upregulation and overextension of actin-based cell protrusions were also involved in hemocyte invasion in mxc(mbn1) larvae. These findings contribute to the understanding of molecular mechanisms underlying mammalian leukemic invasion (Takarada, 2023).

Mutual repression between JNK/AP-1 and JAK/STAT stratifies senescent and proliferative cell behaviors during tissue regeneration

Epithelial repair relies on the activation of stress signaling pathways to coordinate tissue repair. Their deregulation is implicated in chronic wound and cancer pathologies. Using TNF-α/Eiger-mediated inflammatory damage to Drosophila imaginal discs, this study investigate how spatial patterns of signaling pathways and repair behaviors arise. Eiger expression, which drives JNK/AP-1 signaling, was found to transiently arrest proliferation of cells in the wound center and is associated with activation of a senescence program. This includes production of the mitogenic ligands of the Upd family, which allows JNK/AP-1-signaling cells to act as paracrine organizers of regeneration. Surprisingly, JNK/AP-1 cell-autonomously suppress activation of Upd signaling via Ptp61F and Socs36E, both negative regulators of JAK/STAT signaling. As mitogenic JAK/STAT signaling is suppressed in JNK/AP-1-signaling cells at the center of tissue damage, compensatory proliferation occurs by paracrine activation of JAK/STAT in the wound periphery. Mathematical modelling suggests that cell-autonomous mutual repression between JNK/AP-1 and JAK/STAT is at the core of a regulatory network essential to spatially separate JNK/AP-1 and JAK/STAT signaling into bistable spatial domains associated with distinct cellular tasks. Such spatial stratification is essential for proper tissue repair, as coactivation of JNK/AP-1 and JAK/STAT in the same cells creates conflicting signals for cell cycle progression, leading to excess apoptosis of senescently stalled JNK/AP-1-signaling cells that organize the spatial field. Finally, this study demonstrated that bistable separation of JNK/AP-1 and JAK/STAT drives bistable separation of senescent signaling and proliferative behaviors not only upon tissue damage, but also in RasV12, scrib tumors. Revealing this previously uncharacterized regulatory network between JNK/AP-1, JAK/STAT, and associated cell behaviors has important implications for conceptual understanding of tissue repair, chronic wound pathologies, and tumor microenvironments (Jaiswal, 2023).

Long non-coding RNA CR46040 is essential for injury-stimulated regeneration of intestinal stem cells in Drosophila

Long non-coding RNAs (lncRNAs) play important regulatory roles in stem cells self-renewal, pluripotency maintenance and differentiation. Till now, there is very limited knowledge about how lncRNAs regulate intestinal stem cells (ISCs), and lncRNAs mediating ISCs regeneration in Drosophila have yet been characterized. This study identify a lncRNA, CR46040, that is essential for the injury-induced ISCs regeneration in Drosophila. Loss of CR46040 greatly impairs ISCs proliferation in response to tissue damage caused by dextran sulfate sodium (DSS) treatment. This study demonstrates that CR46040 is a genuine lncRNA that has two isoforms transcribed from the same transcription start site, and works in trans to regulate intestinal stem cells. Mechanistically, CR46040 knock-out flies are failed to fully activate JNK, JAK/STAT and HIPPO signaling pathways after tissue damage, which are required for ISCs proliferation after intestinal injury. Moreover, CR46040 knock-out flies are highly susceptible to DSS treatment and enteropathogenic bacteria Erwinia carotovora ssp. carotovora 15 (Ecc15) infection. These findings characterize, for the first time, a lncRNA that mediates damage-induced ISCs proliferation in Drosophila, and provide new insights into the functional links among the long non-coding RNAs, ISCs proliferation and tissue homeostasis (Xu, 2023).

A conserved mechanism for JNK-mediated loss of Notch function in advanced prostate cancer

Dysregulated Notch signaling is a common feature of cancer; however, its effects on tumor initiation and progression are highly variable, with Notch having either oncogenic or tumor-suppressive functions in various cancers. To better understand the mechanisms that regulate Notch function in cancer, Notch signaling was studied in a Drosophila tumor model, prostate cancer-derived cell lines, and tissue samples from patients with advanced prostate cancer. It was demonstrated that increased activity of the Src-JNK pathway in tumors inactivated Notch signaling because of JNK pathway-mediated inhibition of the expression of the gene encoding the Notch S2 cleavage protease, Kuzbanian, which is critical for Notch activity. Consequently, inactive Notch accumulated in cells, where it was unable to transcribe genes encoding its target proteins, many of which have tumor-suppressive activities. These findings suggest that Src-JNK activity in tumors predicts Notch activity status and that suppressing Src-JNK signaling could restore Notch function in tumors, offering opportunities for diagnosis and targeted therapies for a subset of patients with advanced prostate cancer (Wang, 2023).

E2F1 promotes, JNK and DIAP1 inhibit, and chromosomal position has little effect on radiation-induced Loss of Heterozygosity in Drosophila

Loss of Heterozygosity (LOH) can occur when a heterozygous mutant cell loses the remaining wild type allele to become a homozygous mutant. LOH can have physiological consequences if, for example, the affected gene encodes a tumor suppressor. This study used two fluorescent reporters to study mechanisms of LOH induction by X-rays, a type of ionizing radiation (IR), in Drosophila larval wing discs. IR is used to treat more than half of cancer patients, so understanding its effects is of biomedical relevance. IR-induced LOH does not correlate with the chromosomal position of the LOH locus, unlike previously shown for spontaneously occurring LOH. Like spontaneous LOH, however, IR-induced LOH of X-linked loci shows a sex-dependence, occurring predominately in females. A focused genetic screen identified E2F1 as a key promotor of LOH and further testing suggests a mechanism involving its role in cell cycle regulation rather than apoptosis. The QF/QS LOH reporter was combined with QUAS-transgenes to manipulate gene function after LOH induction. This approach identified JNK signaling and apoptosis as key determinants of LOH maintenance. These studies reveal previously unknown mechanisms for generation and maintenance of cells with chromosome aberrations after exposure to IR (Brown, 2023).

N-Acetyltransferase 9 ameliorates Aβ42-mediated neurodegeneration in the Drosophila eye

Alzheimer's disease (AD), a progressive neurodegenerative disorder, manifests as accumulation of amyloid-beta-42 (Aβ42) plaques and intracellular accumulation of neurofibrillary tangles (NFTs) that results in microtubule destabilization. Targeted expression of human Aβ42 (GMR > Aβ42) in developing Drosophila eye retinal neurons results in Aβ42 plaque(s) and mimics AD-like extensive neurodegeneration. However, there remains a gap in understanding of the underlying mechanism(s) for Aβ42-mediated neurodegeneration. To address this gap in information, a forward genetic screen was conducted, and N-acetyltransferase 9 (Mnat9) was identified as a genetic modifier of GMR > Aβ42 neurodegenerative phenotype. Mnat9 is known to stabilize microtubules by inhibiting c-Jun-N- terminal kinase (JNK) signaling. This study found that gain-of-function of Mnat9 rescues GMR > Aβ42 mediated neurodegenerative phenotype whereas loss-of-function of Mnat9 exhibits the converse phenotype of enhanced neurodegeneration. A new neuroprotective function of Mnat9 is proposed in downregulating the JNK signaling pathway to ameliorate Aβ42-mediated neurodegeneration, which is independent of its acetylation activity. Transgenic flies expressing human NAT9 (hNAT9), also suppresses Aβ42-mediated neurodegeneration thereby suggesting functional conservation in the interaction of fly Mnat9or hNAT9 with JNK-mediated neurodegeneration. These studies add to the repertoire of molecular mechanisms that mediate cell death response following accumulation of Aβ42 and may provide new avenues for targeting neurodegeneration (Deshpande, 2023).

FSBP suppresses tumor cell migration by inhibiting the JNK pathway

The main cause of high mortality in cancer patients is tumor metastasis. Exploring the underlying mechanism of tumor metastasis is of great significance for clinical treatments. This study has identified the transcription factor Apt/FSBP is a suppressor for tumor metastasis. In Drosophila wing disc, knockdown of apt is able to trigger cell migration, whereas overexpression of apt hampers scrib-RNAi-induced tumor cell migration. Further studies show that loss of apt promotes cell migration JNK pathway . To investigate the role of FSBP, the homolog of Apt in mammals, Fsbp liver-specific knockout mice were constructed. Knockout of Fsbp in liver does not cause any detectable physiological defects, but predisposes to tumorigenesis on DEN and CCl(4) treatment. In addition, loss of Fsbp accelerates tumor metastasis from liver to diaphragm. Taken together, this study uncovers FSBP is a novel tumor suppressor, and provides it as a considerable drug target for tumor treatment (Song, 2023).

Acetobacter and lactobacillus alleviate the symptom of insulin resistance by blocking the JNK-JAK/STAT pathway in Drosophila melanogaster

The dysregulation of intestinal microbiota is well-known to be one of the main causes of insulin resistance in both vertebrates and invertebrates. Specially, the acetobacter and lactobacillus have been identified as potentially capable of alleviating insulin resistance. However, the molecular mechanism underlying this effect requires further elucidation. In this study, Drosophila was used to delineate how intestinal microbiota disrupts the host intestinal signaling pathway, contributing to insulin resistance. These findings demonstrate that a long-term high-sugar diet leads to a reduction in the general diversity of intestinal microbiota in flies, as well as a marked decrease in the abundances of acetobacter and lactobacillus. Furthermore, symptoms of insulin resistance were alleviated by feeding flies with acetobacter or lactobacillus, indicating that these microorganisms play an essential role in maintaining blood sugar homeostasis in flies. Conversely, when all intestinal microbiota was removed, flies show severe symptoms of insulin resistance, confirming that the critical role of intestinal microbiota in maintaining host blood sugar homeostasis. These studies suggested that the intestinal but not fat body JNK pathway mediates the communication of intestinal microbiota and host insulin pathway. In flies, downregulation of JNK activity alleviates symptoms of insulin resistance by decreasing the activity of the JAK/STAT pathway. However, this offsets the therapeutic effects of supplying flies with acetobacter or lactobacillus, suggesting that the therapeutic function of these microorganisms is based on their interaction with JNK-JAK/STAT axis. Taken together, this study reveals that acetobacter and lactobacillus alleviate insulin resistance symptoms in a JNK-JAK/STAT pathway-dependent manner, indicating the therapeutic potential of probiotic supplementation and regulation of the activities of JNK-JAK/STAT pathway for diabetes control (Meng, 2023).

Silibinin alleviates intestinal inflammation via inhibiting JNK signaling in Drosophila

Inflammatory bowel diseases (IBDs) are characterized by chronic relapsing intestinal inflammation that causes digestive system dysfunction. For years, researchers have been working to find more effective and safer therapeutic strategies to treat these diseases. Silibinin (SIL), a flavonoid compound extracted from the seeds of milk thistle plants, possesses multiple biological activities and is traditionally applied to treat liver diseases. SIL is also widely used in the treatment of a variety of inflammatory diseases attributed to its excellent antioxidant and anti-inflammatory effects. However, the efficacy of SIL against IBDs and its mechanisms remain unclear. This study, using Drosophila melanogaster as a model organism, found that SIL can effectively relieve intestinal inflammation caused by dextran sulfate sodium (DSS). The results suggested that SIL supplementation can inhibit the overproliferation of intestinal stem cells (ISCs) induced by DSS, protect intestinal barrier function, acid-base balance, and intestinal excretion function, reduce intestinal reactive oxygen species (ROS) levels and inflammatory stress, and extend the lifespan of Drosophila. Furthermore, this study demonstrated that SIL ameliorates intestinal inflammation via modulating the c-Jun N-terminal kinase (JNK) signaling pathway in Drosophila. This research aims to provide new insight into the treatment of IBDs (Yan, 2023).

Extracellular matrix protein N-glycosylation mediates immune self-tolerance in Drosophila melanogaster

In order to respond to infection, hosts must distinguish pathogens from their own tissues. This allows for the precise targeting of immune responses against pathogens and also ensures self-tolerance, the ability of the host to protect self tissues from immune damage. One way to maintain self-tolerance is to evolve a self signal and suppress any immune response directed at tissues that carry this signal. This study characterizes the Drosophila tuSz mutant strain, which mounts an aberrant immune response against its own fat body. This study demonstrates that this autoimmunity is the result of two mutations: 1) a mutation in the Glucosidase 1/GCS1 gene that disrupts N-glycosylation of extracellular matrix proteins covering the fat body, and 2) a mutation in the Drosophila Janus Kinase ortholog that causes precocious activation of hemocytes. Data indicate that N-glycans attached to extracellular matrix proteins serve as a self signal and that activated hemocytes attack tissues lacking this signal. The simplicity of this invertebrate self-recognition system and the ubiquity of its constituent parts suggests it may have functional homologs across animals (Mortimer, 2021).

Models describing the necessity for two independent signals in fly encapsulation responses. (A) Homeostasis is maintained in naive wild-type larvae. (B) In tuSz¹ mutant larvae, immune cells are inappropriately activated by JAK-STAT pathway activation due to the hop^Sz gain-of-function mutation. The loss of protein N-glycosylation in posterior fat body tissue due to the GCS1^Sz mutation leads to loss of self-tolerance and tissue encapsulation. (C) In the model of self-tolerance described in a previous study, the coupled phenotypes of loss of cell integrity and loss of ECM integrity are sufficient to disrupt self-tolerance. (D) Immune cells are activated following parasitoid wasp infection, presumably due to the wound-mediated activation of JAK-STAT signaling. SAMP-presenting host tissues are protected from encapsulation, and wasp eggs may be targeted for encapsulation because they are missing the ECM N-glycosylation SAMP (Mortimer, 2021).

References

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

Brown, J. and Su, T. T. (2023). E2F1 promotes, JNK and DIAP1 inhibit, and chromosomal position has little effect on radiation-induced Loss of Heterozygosity in Drosophila. bioRxiv. PubMed ID: 37214983

Brown, J., Su, T. T. (2024). E2F1, DIAP1, and the presence of a homologous chromosome promote while JNK inhibits radiation-induced loss of heterozygosity in Drosophila melanogaster. Genetics, 226(1) PubMed ID: 37874851

Crucianelli, C., Jaiswal, J., Vijayakumar Maya, A., Nogay, L., Cosolo, A., Grass, I. and Classen, A. K. (2022). Distinct signaling signatures drive compensatory proliferation via S-phase acceleration. PLoS Genet 18(12): e1010516. PubMed ID: 36520882

Datta, I. and Bangi, E. (2023). Senescent cells and macrophages cooperate through a multi-kinase signaling network to promote intestinal transformation in Drosophila. bioRxiv. PubMed ID: 37292988

Deshpande, P., Chimata, A. V., Snider, E., Singh, A., Kango-Singh, M. and Singh, A. (2023). N-Acetyltransferase 9 ameliorates Aβ42-mediated neurodegeneration in the Drosophila eye. Cell Death Dis 14(7): 478. PubMed ID: 37507384

D'Souza, L. C., Dwivedi, S., Raihan, F., Yathisha, U. G., Raghu, S. V., Mamatha, B. S. and Sharma, A. (2022). Hsp70 overexpression in Drosophila hemocytes attenuates benzene-induced immune and developmental toxicity via regulating ROS/JNK signaling pathway. Environ Toxicol 37(7): 1723-1739. PubMed ID: 35301792

Farrell, L., Puig-Barbe, A., Haque, M. I., Amcheslavsky, A., Yu, M., Bergmann, A. and Fan, Y. (2022). Actin remodeling mediates ROS production and JNK activation to drive apoptosis-induced proliferation. PLoS Genet 18(12): e1010533. PubMed ID: 36469525

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

Garcia-Arias, J. M., Pinal, N., Cristobal-Vargas, S., Estella, C. and Morata, G. (2023). Lack of apoptosis leads to cellular senescence and tumorigenesis in Drosophila epithelial cells. Cell Death Discov 9(1): 281. PubMed ID: 37532716

Jaiswal, J., Egert, J., Engesser, R., Peyroton, A. A., Nogay, L., Weichselberger, V., Crucianelli, C., Grass, I., Kreutz, C., Timmer, J. and Classen, A. K. (2023). Mutual repression between JNK/AP-1 and JAK/STAT stratifies senescent and proliferative cell behaviors during tissue regeneration. PLoS Biol 21(5): e3001665. PubMed ID: 37252939

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

Karkali, K., Saunders, T. E., Panayotou, G. and Martin-Blanco, E. (2023). JNK signaling in pioneer neurons organizes ventral nerve cord architecture in Drosophila embryos. Nat Commun 14(1): 675. PubMed ID: 36750572

Kim, H. K., Kim, C. J., Jang, D. and Lim, D. H. (2023). MicroRNA miR-274-5p supresses Found-in-Neurons associated with Melanotic mass Formation and Developmental Growth in Drosophila. Insects 14(8). PubMed ID: 37623419

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

Kumar, A. and Baker, N. E. (2022). The CRL4 E3 ligase Mahjong/DCAF1 controls cell competition through the transcription factor Xrp1, independently of polarity genes. Development 149(22). PubMed ID: 36278853

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

Li, C., Zhu, X., Sun, X., Guo, X., Li, W., Chen, P., Shidlovskii, Y. V., Zhou, Q., Xue, L. (2023). Slik maintains tissue homeostasis by preventing JNK-mediated apoptosis. Cell division, 18(1):16 PubMed ID: 37794497

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.

Liu, W., Lim, K. L. and Tan, E. K. (2022). Intestine-derived α-synuclein initiates and aggravates pathogenesis of Parkinson's disease in Drosophila. Transl Neurodegener 11(1): 44. PubMed ID: 36253844

Meng, Q., Xu, Y., Li, Y. and Wang, Y. (2022). Novel studies on Drosophila melanogaster model reveal the roles of JNK-Jak/STAT axis and intestinal microbiota in insulin resistance. J Drug Target: 1-8. PubMed ID: 36343203

Meng, Q., Li, Y., Xu, Y., Wang, Y. (2024). Acetobacter and lactobacillus alleviate the symptom of insulin resistance by blocking the JNK-JAK/STAT pathway in Drosophila melanogaster. Biochimica et biophysica acta Molecular basis of disease, 1870(1):166901 PubMed ID: 37774935

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

Mortimer, N. T., Fischer, M. L., Waring, A. L., Kr, P., Kacsoh, B. Z., Brantley, S. E., Keebaugh, E. S., Hill, J., Lark, C., Martin, J., Bains, P., Lee, J., Vrailas-Mortimer, A. D. and Schlenke, T. A. (2021). Extracellular matrix protein N-glycosylation mediates immune self-tolerance in Drosophila melanogaster. Proc Natl Acad Sci U S A 118(39). PubMed ID: 34544850

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

Prasad, D., Illek, K., Fischer, F., Holstein, K. and Classen, A. K. (2023). Bilateral JNK activation is a hallmark of interface surveillance and promotes elimination of aberrant cells. Elife 12. PubMed ID: 36744859

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

Sakakibara, Y., Yamashiro, R., Chikamatsu, S., Hirota, Y., Tsubokawa, Y., Nishijima, R., Takei, K., Sekiya, M. and Iijima, K. M. (2023). Drosophila Toll-9 is induced by aging and neurodegeneration to modulate stress signaling and its deficiency exacerbates tau-mediated neurodegeneration. iScience 26(2): 105968. PubMed ID: 36718365

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

Song, F., Zhang, W., Li, X., Chen, X., Yuan, X., Jiang, M., Zhao, Y., Liu, Q. and Zhou, Z. (2023). FSBP suppresses tumor cell migration by inhibiting the JNK pathway. iScience 26(4): 106440. PubMed ID: 37035004

Sriskanthadevan-Pirahas, S., Tinwala, A. Q., Turingan, M. J., Khan, S., Grewal, S. S. (2023). Mitochondrial metabolism in Drosophila macrophage-like cells regulates body growth via modulation of cytokine and insulin signaling. Biol Open, 12(11) PubMed ID: 37850733

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

Takarada, K., Kinoshita, J. and Inoue, Y. H. (2023). Ectopic expression of matrix metalloproteinases and filopodia extension via JNK activation are involved in the invasion of blood tumor cells in Drosophila mxc mutant. Genes Cells. PubMed ID: 37615261

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

Valencia-Exposito, A., Gomez-Lamarca, M. J., Widmann, T. J. and Martin-Bermudo, M. D. (2022). Integrins Cooperate With the EGFR/Ras Pathway to Preserve Epithelia Survival and Architecture in Development and Oncogenesis Front Cell Dev Biol 10: 892691. PubMed ID: 35769262

Wang, C. W., Clemot, M., Hashimoto, T., Diaz, J. A., Goins, L. M., Goldstein, A. S., Nagaraj, R., Banerjee, U. (2023). A conserved mechanism for JNK-mediated loss of Notch function in advanced prostate cancer. Science signaling, 16(810):eabo5213 PubMed ID: 37934809

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

Wang, Z., Xia, X., Li, J. and Igaki, T. (2022). Tumor elimination by clustered microRNAs miR-306 and miR-79 via noncanonical activation of JNK signaling. Elife 11. PubMed ID: 36222503

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

Worley, M. I., Everetts, N. J., Yasutomi, R., Chang, R. J., Saretha, S., Yosef, N. and Hariharan, I. K. (2022). Ets21C sustains a pro-regenerative transcriptional program in blastema cells of Drosophila imaginal discs. Curr Biol 32(15): 3350-3364. PubMed ID: 35820420

Xu, Q., Liu, J., Du, X., Xue, D., Li, D. and Bi, X. (2023). Long non-coding RNA CR46040 is essential for injury-stimulated regeneration of intestinal stem cells in Drosophila. Genetics. PubMed ID: 36930573

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

Yan, L., Zhou, J., Yuan, L., Ye, J., Zhao, X., Ren, G., Chen, H. (2023). Silibinin alleviates intestinal inflammation via inhibiting JNK signaling in Drosophila. Frontiers in pharmacology, 14:1246960 PubMed ID: 37781701

Zhang, W., Wang, D., Si, J., Jin, L. H. and Hao, Y. (2023). Gbb Regulates Blood Cell Proliferation and Differentiation through JNK and EGFR Signaling Pathways in the Drosophila Lymph Gland Cells 12(4). PubMed ID: 36831328

date revised: 10 July 2024

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