REVIEW doi: 10.1111/sji.12170 ..................................................................................................................................................................

JAK/STAT Pathway in Drosophila Immunity H. Myllyma¨ki* & M. Ra¨met*†‡§

Abstract *Laboratory of Experimental Immunology, BioMediTech, University of Tampere, Tampere, Finland; †Department of Pediatrics, Tampere University Hospital, Tampere, Finland; ‡Department of Pediatrics, Medical Research Center Oulu, University of Oulu, Oulu, Finland; and §Department of Children and Adolescents, Oulu University Hospital, Oulu, Finland

Received 18 February 2014; Accepted in revised form 21 March 2014

The Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling pathway controls multiple biological processes in metazoan development and tissue homoeostasis. This creates a need for tight regulatory mechanisms to ensure proper responses. The core components of the pathway, as well as many of the regulatory molecules, are conserved in evolution and thus share similarities in organisms such as humans and fruit flies. Therefore, the fruit fly provides an amenable model system for elucidating the in vivo roles of the JAK/STAT pathway and its regulators, which are challenging to demonstrate in mammalian systems. This review will focus on describing recent advances in understanding the importance of JAK/STAT signalling in Drosophila immunity.

Correspondence to: M. Ra¨met, Laboratory of Experimental Immunology, BioMedi Tech, University of Tampere, FI-33014 Tampere, Finland. E-mail: [email protected]

Introduction The importance of interferons and Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling in the regulation of human immunity has been recognized for decades [1]. This has led to intense research on the biochemistry, genetics and biological roles of JAK/ STAT-mediated responses in immunity. For example, JAK/STAT signalling has been associated with several aspects of the innate immune system, including the control of inflammatory responses and wound repair, as well as the activation of neutrophils and macrophages. Furthermore, JAK/STAT signalling has been found to be involved in the differentiation of T and B cells of the adaptive immune system [2]. Correspondingly, mutations that affect components of the pathway and situations that otherwise disturb signalling have been linked with several human diseases, including autoimmune disorders, such as rheumatoid arthritis, inflammatory bowel disease, psoriasis and Crohn’s disease [3]. Loss-of-function mutations in pathway components cause susceptibility to various infections or even the autosomal recessive form of severe combined immunodeficiency (SCID) [4], while hyperactivating mutations are found in many cancers, such as lymphomas [5, 6]. In humans, the four JAKs (JAK1, JAK2, JAK3 and TYK2) and seven STATs (STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6) mediate responses to a number of cytokines, such as interferons, interleukins and colony-stimulating factors. The canonical model of JAK/STAT signalling makes the molecular

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events in the signalling pathway appear rather simple. The pathway is, however, very complex in an actual biological context due to the number of cytokines that can activate it, and the ability of the JAKs and STATs to form homo- and heterodimers and associate with multiple transcription factors and co-activators [5]. As the JAK/ STAT pathway is highly conserved in evolution, the fruit fly Drosophila melanogaster provides an amenable and more robust model system, with less redundancy for studying especially the in vivo roles of molecules that regulate JAK/ STAT signalling [7].

Activation and regulation of the JAK/STAT pathway in Drosophila The Drosophila JAK/STAT pathway has the same essential signalling components as in mammals, but there is less redundancy (Fig. 1). In Drosophila, the known JAK/STAT pathway ligands consist of only three cytokine-like proteins called unpaired (upd) [8], upd2 [9, 10] and upd3 [11, 12]. The genes coding for the upd molecules are clustered in the Drosophila genome on the X chromosome and have no obvious homologues outside the Drosophila species, but share some similarity with the vertebrate leptins [8, 10]. Upd is associated with the extracellular matrix [8], while upd2 is soluble and able to diffuse freely. All three upd molecules are induced locally in response to tissue damage such as wounding, upd3 expression is induced in adult haemocytes upon bacterial challenge, and both upd2 and upd3 are induced in response to viral

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Figure 1 The Drosophila JAK/STAT pathway. The core components of the pathway have been conserved in evolution and signalling is thought to follow the canonical mode. Several positive and negative regulators have been identified (see text for details).

infections, suggesting that multiple regulatory mechanisms control their expression [11–14]. All three upd molecules signal – and putatively bind – via a single receptor, Domeless (Dome), which shares functional and sequence similarity with the mammalian cytokine class I receptors, such as the IL-6 receptor, both of which have the extracellular fibronectin type III domain and a cytokine-binding module (CBM) [15, 16]. Moreover, Drosophila has a single JAK, hopscotch (hop) [17, 18], and one STAT transcription factor, Stat92E [19, 20]. These proteins share most sequence similarity with JAK2 and STAT5, which are required for mediating responses to IL3, IL-5 and IFN-c, several haematopoietic growth factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO), as well as the mitogens growth hormone (GH) and prolactin [5, 21]. Drosophila JAK/STAT activation is thought to follow the canonical signalling model similar to that described in mammals. According to the model, the binding of a cytokine to its receptor induces receptor dimerization and activation of the JAKs that are constitutively associated with the cytoplasmic tail of the receptor. Activated JAKs phosphorylate each other and specific tyrosine residues on the cytoplasmic part of the receptor, which act as docking sites for the Src homology 2 (SH2) domains of STAT molecules. Also, the STATs are tyrosine phosphorylated by JAKs, which allows them to form dimers and translocate into the nucleus, where they bind the promoters of their target genes [5]. In Drosophila, Stat92E is phosphorylated on Tyr-704, and the consensus binding sequence is TTCCCGGAA [16, 20, 22]. As the JAK/STAT pathway is involved in multiple biological processes, signalling needs to be strictly controlled at different levels of the cascade in both Drosophila and mammals. Like the pathway components, many of the

regulators show high conservation in evolution [23]. In addition to homology-based identification of regulators of the Drosophila JAK/STAT pathway, intense investigation has led to the discovery of several new regulators by experimental methods, such as forward and reverse genetic screening [24–28]. In mammals, JAK/STAT signalling is regulated at the receptor level by the membrane-spanning signal transducer protein gp130, which is used, for example, by members of the IL-6 receptor family. gp130 mediates signalling by forming complexes with cytokine receptors that have bound their ligands and by associating with JAKs via its cytoplasmic domain. On the other hand, the soluble form of gp130 is able to sequester ligands and therefore inhibit signalling [29]. In Drosophila, a non-signalling protein that resembles gp130 and the JAK/STAT pathway receptor Dome is also found to regulate signalling activity. Eye transformer (ET, also called latran) is located next to Dome in the Drosophila genome, and the two molecules also share similarity in structure, but ET has a short cytoplasmic tail that lacks the Stat92E-binding site found on the cytoplasmic part of Dome. ET is associated with the receptor complex, interacting with both Dome and hop, and it seems plausible that ET inhibits intracellular signalling [27, 30]. However, the function of the cytokine-binding modules found on ET’s extracellular part remains elusive, as well as the exact nature of the interaction of ET and Dome and the composition and stoichiometry of the complex they form. Negative feedback loops are a classical mechanism for regulating signalling pathways. In mammalian JAK/STAT signalling, this task is fulfilled by the suppressor of cytokine signalling (SOCS) proteins, for which humans have eight genes (SOCS1-7 and CIS) [31, 32]. This mechanism is also found in Drosophila, whose genome codes for three members of the family, Socs16D, Socs36E and Socs44A. Socs16D has no known role in the regulation of the JAK/STAT pathway, while Socs44A appears to regulate JAK/STAT signalling to some extent, even though it is not a transcriptional target of Stat92E [33, 34]. The main negative feedback loop regulator of these is Socs36E, which is most related to mammalian SOCS5 and is strongly induced by JAK/STAT signalling [35, 36]. Like the mammalian SOCSs, Socs36E has a central SH2 domain and a C-terminal SOCS box, which in mammals bind phosphorylated tyrosine residues and interact with the enzymes of the ubiquitinating machinery, respectively [32]. Recently, the molecular mechanism of Socs36E inhibition was characterized in more detail, showing that Socs36E indeed uses both of these two activities to downregulate JAK/STAT signalling. First, Socs36E functions via its SOCS box by a mechanism involving the E3 ubiquitin ligase elongin–cullin–SOCS (ECS) complex. The Drosophila ESC proteins elongin B, elongin C and cullin-5 act to regulate the endocytic trafficking and

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lysosomal degradation of Dome, thereby affecting receptor levels [37, 38]. In addition, Socs36E is able to downregulate both the basal and ligand-induced JAK/STAT pathway activity. This downregulation takes place via an independent mechanism that requires the SH2 domain and the Nterminal part of Socs36E and its interaction with Dome and thus possibly inhibits receptor phosphorylation by hop [37]. Another negative regulator that functions at the kinase level is the protein tyrosine phosphatase Ptp61F, which was identified in two separate RNAi screens [24, 26]. Ptp61F is a transcriptional target of the JAK/STAT pathway and thus also functions via a negative feedback loop, presumably by targeting activated hop (and possibly also Stat92E) for deactivation [24, 26]. The activity, location and DNA-binding ability of Stat92E are also subject to regulation. The Drosophila homologues of Ras-like guanine nucleotide-binding protein 3 (RanBP3) and RanBP10 negatively regulate JAK/ STAT signalling by controlling the signal-dependent nuclear transport of Stat92E [24]. Stat92E DNA binding is positively and negatively regulated by several molecules. The human CCR4-NOT transcription regulation complex subunit 4 (CNOT4) appears to regulate STAT-mediated gene responses, and also the Drosophila homologue, called Not4, is needed for proper Stat92E DNA binding [28]. In mammals, protein inhibitors of activated STATs (PIAS) molecules have been shown to bind tyrosine-phosphorylated STAT dimers (but not unphosphorylated or monomeric STATs) and thereby block their binding to DNA [39]. Drosophila has a single Pias gene, whose importance in the negative regulation of the JAK/STAT pathway has been studied in vivo and shows that the correct dPias/ Stat92E gene ratio is crucial for blood cell and eye development. dPIAS was also shown to interact with activated Stat92E, suggesting a similar molecular function with the mammalian homologues [40]. It has been shown that the mammalian PIAS proteins can function as E3-type ligases for small ubiquitin-like modifier (SUMO) molecules on various target proteins, including STAT1, and that STAT activity is inhibited by sumoylation [41]. Moreover, it has also been shown that the Drosophila Stat92E activity is negatively regulated by sumoylation on Lys187 [42], but it remains to be studied whether dPIAS is involved in Stat92E sumoylation. Ken & barbie (Ken) is an ortholog of the mammalian proto-oncogene B-cell lymphoma 6 (BCL6), with an Nterminal BTB/POZ domain and three C-terminal C2H2 zinc finger motifs. BCL6 can act as a repressor of STAT6dependent target gene expression in cell culture [43]. In Drosophila, a subset of Stat92E target gene promoters contain Ken binding sites that overlap with the sites of Stat92E, subjecting these genes to downregulation by Ken, while expression of the other Stat92E target genes remains unaffected [24, 44]. The Drosophila homologue of the bromo-domain-containing protein BRWD3 was found to

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regulate Drosophila JAK/STAT signalling positively in vivo, but the molecular mechanism remains elusive [26]. The same study also identified a gene named Diedel as a potential negative regulator of the JAK/STAT pathway, and the crystal structure of the corresponding cysteine-rich extracellular protein was recently solved [26, 45].

JAK/STAT signalling in a biological context The JAK/STAT pathway controls several biological processes in both Drosophila embryos and adults, including embryonic patterning [15, 19, 46], formation of the wing and eye [20, 25] and maintenance of stem cells in their niches [47–49]. Therefore, loss-of-function alleles of the Drosophila JAK/STAT pathway core components are embryonic lethal or cause varying developmental defects [8, 15, 18, 20]. The JAK/STAT pathway also directly contributes to immune and stress responses by activating infection-induced genes [23, 50]. The humoural immune response in Drosophila culminates in the production and secretion of a patrol of antimicrobial peptides by the cells of the fat body, which is mainly controlled by the Toll and Imd pathways (reviewed in [51, 52]). However, the JAK/ STAT pathway is responsible for the expression of several additional immune-related proteins, including cytokines and stress response proteins in the fat body. This is induced by the JAK/STAT pathway ligand upd3, which is produced by the haemocytes in response to septic injury or other stress, such as heat-shock or dehydration [53]. One such group of stress-induced genes is the Turandot (Tot) family, whose expression is dependent on both the JAK/ STAT and Imd pathways [27, 54]. Tot proteins are found in large quantities in the haemolymph, but their exact function is not known [11, 53, 54]. In addition, Drosophila JAK/STAT signalling controls haemocyte proliferation and differentiation [55, 56] and tissue repair (Fig. 3). JAK/STAT signalling in Drosophila haematopoiesis and cellular immunity

Drosophila has an open circulatory system where the haemolymph floods in the body cavity (called haemocoel). Three types of haemocytes can be found: plasmatocytes, lamellocytes and crystal cells. The most abundant of the blood cells are the plasmatocytes, macrophage-like cells found in both Drosophila larvae and adults as a circulating and sessile population [57]. Their main function is to carry out the phagocytosis of pathogens and apoptotic host cells [58]. The two rarer blood cell types in Drosophila are lamellocytes and crystal cells, which are responsible for the encapsulation of foreign invaders that are too large to be phagocytosed, and melanization reactions, respectively [56, 59]. During Drosophila larval development, the haemocytes are generated in an organ called the lymph gland. The lymph gland consists of three zones with distinctive

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functions: the medullary zone, the cortical zone and the posterior signalling centre (PSC) [60, 61]. The PSC functions as the haematopoietic niche and regulates haematopoietic stem cell maintenance and development [62]. In a normal, healthy animal, there are no or very few lamellocytes [57, 63]. However, Drosophila is naturally infected by several parasitic wasps, such as Leptopilina boulardi, that lay their eggs in the fly larvae (Fig. 2). A parasitic infestation is recognized by the circulating plasmatocytes, which can attach to the surface of the egg. The attached plasmatocytes can differentiate into lamellocytes and thereby initiate the encapsulation reaction [64, 65]. They also appear to communicate with the lymph gland, but the signals are not fully understood. It appears that the EGFR ligand Spitz is involved in signalling from the lymph gland to circulating and sessile haemocytes for lamellocyte differentiation [62, 63, 66, 67]. Mature lamellocytes are large, flattened cells that attack the parasite in order to encapsulate it [59]. The cell capsule is then melanized by crystal cells, which induce a phenoloxidase cascade leading to the synthesis of melanin and reactive oxygen species [57]. There is variation in the efficiency of both the wasp invasion and the Drosophila defence, depending on the wasp species [68]. Nevertheless, successive parasitism leads to hatching of the wasp larva, which then grows inside the Drosophila larval host, and ultimately ecloses as an adult wasp from the pupa. On the other hand, full encapsulation of the wasp egg by the Drosophila host prevents it from hatching and allows the survival of the fly. The exact signalling events and mechanisms needed for the induction of lamellocyte differentiation in the lymph

A

gland are not fully understood. However, JAK/STAT signalling has been shown to play an important role in the process. Generally, loss-of-functions in JAK/STAT pathway components result in an impaired encapsulation capacity and reduced lymph gland immune responsiveness [56]. On the other hand, aberrant activation of the JAK/ STAT (or Toll) pathway in the absence of a parasitic infection induces premature lamellocyte differentiation. This leads to lamellocyte accumulation and the subsequent formation of melanotic tumours due to host material becoming ‘encapsulated’ by these cells [55, 56, 69]. Genetic examples of such a situation include hyperactive alleles of Drosophila hop, for example the temperaturesensitive tumorous-lethal (hopTum-l). The same phenotype results from a number of other alleles that hyperactivate JAK/STAT signalling, as well as from the overexpression of wild-type hop in the lymph gland [55, 70, 71]. The mammalian equivalent is a gain-of-function mutation in human JAK2 that causes myeloproliferative disorders [3]. However, in the lymph gland, the upd3 secreted by the PSC maintains a basal level of JAK/STAT signalling in prohemocytes in the medullary zone to keep them in a nondifferentiating state. In order to induce lamellocyte differentiation in response to parasitic infection, JAK/ STAT signalling in the PSC is tuned down by the downregulation of Dome and upd3 expression [30, 62]. The residual JAK/STAT signalling activity in the prohemocytes is switched off by ET, whose expression is upregulated in response to wasp parasitization [30, 69] (Fig. 2). Therefore, maintaining the homoeostasis between haemocyte proliferation and differentiation appears to be complex and requires a carefully controlled level of JAK/STAT signalling [57].

B

Figure 2 JAK/STAT signalling in Drosophila larval haematopoiesis. JAK/STAT signalling regulates the pro- liferation and differentiation of haemocytes. JAK/STAT activity is needed for lamellocyte differentiation in response to a parasitic infestation (A), but aberrant activity leads to premature lamellocyte differentiation and the formation of melanotic tumours (B).

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Drosophila is emerging as a promising model to study the link between cancer, tissue damage and immunity, and haemocytes and JAK/STAT signalling have been shown to play a role in regulating these processes [72]. Using a genetic model in which tumours are generated in the eye imaginal discs of Drosophila larvae, Pastor-Pareja et al. showed that the disruption of the basement membrane of the discs by either wounding or the growing tumour is detected by circulating plasmatocytes. Plasmatocytes adhere to the wound or tumours and help restricting tumour growth. In addition, the JNK signalling pathway is induced in the tumour cells, leading to the secretion of the upd cytokines. Activated JAK/ STAT signalling generates an amplification loop inducing a systemic response in the haemocytes and fat body, which eventually increases haemocyte proliferation [13]. However, in addition to initiating the antitumour response, JAK/ STAT signalling also appears to contribute to tumour growth by cooperating with oncogenic pathways (Ras in this model) and promoting cell proliferation [73], reminiscent of the situation in humans [74]. Given the conservation of the components involved in these processes, Drosophila is likely to provide novel information about innate immune system and cancers, which may lead to better understanding of human malignancies [13, 72]. JAK/STAT pathway in viral response

In addition to bacteria, fungi and parasites, insects are infected by several viruses and are vectors for many viruses that cause severe human diseases, such as Dengue virus and West Nile virus, therefore creating an important public health concern. Drosophila is naturally infected by more than 25 RNA viruses, including Drosophila C virus (DCV) and Nora virus, and can be experimentally infected with many others, including Cricket paralysis virus and Flock House virus [75–78]. Broadly, the Drosophila viral defence contains two arms: the RNAi machinery that uses Dicer for the recognition of viral dsRNA and Argonaute to restrict viral gene expression, together with a small RNA silencing pathway called the piwi-interacting (pi) RNA pathway. The RNAi-based mechanisms appear to provide a robust defence mechanism against many viruses [79]. The second arm relies on an inducible response dependent on the Toll, Imd and JAK/STAT signalling pathways [80–83]. The set of genes induced by these pathways are distinct from the genes induced by bacterial or fungal infections and also more specific to the virus, indicating that this type of response is tailored according to the infection [14, 83]. The JAK/STAT pathway has been shown to contribute to the innate immune response against viruses in mammals, and its importance in the Drosophila viral response has also been established. The ‘traditional’ JAK/STAT pathway target genes, such as TotM, as well as upd2 and upd3, are induced by multiple viruses, including Flock House virus, Vesicular stomatitis virus and Drosophila X

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virus [14]. Many genes induced by DCV contain STATbinding sites in their promoter regions, and their activation is dependent on the JAK/STAT pathway. In addition, deficiencies in the pathway activation result in increased DCV viral loads and higher mortality. The target genes of the virus-induced JAK/STAT pathway also include some virus-specific ones, such as virus-induced RNA-1 (vir-1), although its function is not known [83] (Fig. 3). Importantly, the JAK/STAT pathway is needed for restricting the Dengue virus in the mosquito Aedes aegypti [84]. The mechanism of viral detection and the induction of the effector signalling pathways in Drosophila and other insects remain rather elusive. An indirect activation mechanism has been suggested for the JAK/STAT pathway, where stress signals sent by infected and damaged cells would be recognized by surrounding uninfected cells. dsRNA itself does not trigger a viral response in Drosophila, but it has been recently shown that recognition of virus-derived dsRNA by the amino terminal DExD/H-box helicase domain of Dicer-2 induces the expression of a secreted protein called vago by a mechanism independent of the other RNAi pathway components [85]. Vago is shown to be antiviral against DCV in Drosophila [86] and in West Nile virus-infected Culex mosquito cells. Moreover, the JAK/STAT pathway induction by vago appears to be independent of Dome, suggesting that an alternative receptor might exist (Fig. 3). Nevertheless, vago provides a link between the RNAi pathway and the JAK/STAT pathway and shows that even though vago appears to be insect specific, it might act as a cytokine that resembles the interferon system in mammals [85]. JAK/STAT pathway in gut immunity

Due to a microbe-enriched diet, the Drosophila gut harbours substantial amounts of potentially pathogenic bacteria, and therefore, the epithelial cells of the gut need to be able to both provide a barrier for preventing a systemic infection and establish antimicrobial activity. In recent years, Drosophila has indeed proved to be a valuable model for studying gut mucosal immunity and homoeostasis. In mammals, this is largely mediated by the innate immunity through mechanisms that are not fully understood, but are likely to be highly conserved in evolution [87]. The antimicrobial arm of the Drosophila gut immunity relies mainly on the DUOX and the Imd pathways and is shown to involve several regulatory mechanisms to avoid massive immune reactions against commensal microbes [88–92]. In addition, the cell damage caused by a pathogenetic infection, for example by Erwinia carotovora carotovora or Serratia marcescens, activates the JAK/STAT pathway [93, 94] (Fig. 3). JAK/STAT signalling contributes to the antimicrobial defence in the gut by inducing the expression of a subset of antimicrobial peptides, such as drosomycin-like peptide (dro3), but this induction is

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A

C

Figure 3 The physiological roles of JAK/ STAT signalling in Drosophila immunity. In the adult fly, JAK/STAT signalling contributes to the systemic immune response (A), the viral response (B), and is needed for the regeneration of the gut epithelium in intestinal infections (C).

pathogen-specific and likely takes place indirectly via the recognition of cell damage rather than the pathogen [93]. Therefore, the major role of JAK/STAT signalling, together with the JNK and DUOX pathways, seems to be in maintaining gut homeostasis during infection by regulating epithelial renewal mechanisms, in particular, to promote cell proliferation in response to bacterial infections [95, 96]. This is presumably induced by upd3 that is secreted by damaged gut cells, mostly enterocytes. Under normal conditions, JAK/STAT (and JNK) activity appears to be important for the proper differentiation of the intestinal stem cell progeny, and activation of the signalling pathway through the expression of hopTum-l or upd3 is sufficient to induce gut renewal even in the absence of infection. The basal gut renewal is stimulated by indigenous gut microbiota [95]. Moreover, upon an E. carotovora infection, flies that are unable to mount proper epithelial renewal have disrupted gut morphology and an increased susceptibility for infections [95], while enhanced JAK/ STAT pathway activity obtained by knocking down the negative regulator ET improves survival upon infection with S. marcescens [27]. Corresponding changes in resistance are also seen when other JAK/STAT pathway components and regulators are knocked down [94].

Conclusions and future perspectives The Drosophila JAK/STAT pathway has been intensely studied both in vitro and in vivo with forward and reverse genetic methods. In addition to homology-based studies, numerous genetic screens have led to the identification of multiple putative regulators [24–28, 97]. However, the picture is far from complete as the overlap between

candidate genes identified in different screens varies [26, 98], and the functional mechanisms of even genes with verified in vivo roles remain partially elusive. In addition, the nature of the inducible viral response in Drosophila is not thoroughly understood and, due to its importance to global human health, needs more studying. JAK/STAT signalling plays an important role in multiple immune and stress-related processes, and its proper function and regulation are crucial for the host. However, the activation of the pathway appears indirect in many cases, as it can be triggered by cell death and stress signals rather than the actual pathogens. Therefore, an effective immune response and tissue recovery requires crosstalk and cooperation with other signalling pathways, such as the Toll and Imd pathways in pathogen recognition and the production of antimicrobial peptides, and the EGFR, Hippo and JNK pathways for stress signalling and tissue repair [99]. In addition, communication between different host tissues and cell types is needed for said responses. Drosophila is emerging as a promising model for studying these signalling networks on a whole-organism level.

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