A tissue communication network coordinating innate immune ......hemocyte recruitment to detached and torn muscles, and the activation of both JAK-STAT and Toll signaling. Importantly,

RESEARCH ARTICLE

A tissue communication network coordinating innate immuneresponse during muscle stressNicole Green, Justin Walker, Alexandria Bontrager, Molly Zych and Erika R. Geisbrecht*

ABSTRACTComplex tissue communication networks function throughout anorganism’s lifespan to maintain tissue homeostasis. Using thegenetic model Drosophila melanogaster, we have defined a networkof immune responses that are activated following the inductionof muscle stresses, including hypercontraction, detachment andoxidative stress. Of these stressors, loss of the genes that causemuscle detachment produced the strongest levels of JAK-STATactivation. In one of these mutants, fondue (fon), we also observehemocyte recruitment and the accumulation of melanin at muscleattachment sites (MASs), indicating a broad involvement of innateimmune responses upon muscle detachment. Loss of fon results inpathogen-independent Toll signaling in the fat body and increasedexpression of the Toll-dependent antimicrobial peptide Drosomycin.Interestingly, genetic interactions between fon and various Tollpathway components enhance muscle detachment. Finally, we showthat JAK-STAT and Toll signaling are capable of reciprocal activation inlarval tissues. We propose a model of tissue communication for theintegration of immune responses at the local and systemic level inresponse to altered muscle physiology.

KEY WORDS: Drosophila, Innate immunity, Tissue communication,Muscle

INTRODUCTIONIn insects, the detection of foreign molecules activates a robustimmune signaling cascade coupled to the production of biologicaloutputs and cellular activities that minimize damage to the host.Innate immunity can be broken down into the humoral arm, whichuses signaling pathways for antimicrobial peptide (AMP) andtarget gene expression, and the cellular arm, which regulates themobilization of hemocytes and melanin production (Royet et al.,2003). Drosophila possesses three types of blood cells: hemocytes,migratory plasmatocytes similar to macrophages; lamellocytes forpathogen encapsulation; and crystal cells, which are crucial forreleasing molecules essential for melanization (Evans and Wood,2014; Wang et al., 2014). Depending on the type of infection,signaling during pathogen challenge proceeds through the canonicalToll (Tl) and immune deficiency (Imd) pathways (Lemaitre et al.,1997). Both of these signal transduction pathways require bindingof an extracellular ligand to transmembrane receptors to activate aseries of intracellular events that lead to the nuclear translocation ofNF-κB transcription factors, Dorsal (Dl) and/or Dif, or Relish (Rel),

respectively (Bergmann et al., 1996; De Gregorio et al., 2002; Hetruand Hoffmann, 2009; Reach et al., 1996; Stöven et al., 2000). Oncein the nucleus, these transcription factors induce the expression ofAMPs and other immune-responsive genes. How these individualaspects of the innate immune response are initiated and integrated topreserve the function of tissues remains an important question inanimal physiology.

Recently, work in model organisms has highlighted novelrelationships between muscle tissue and activation of the innateimmune response. During parasitoid wasp infections, JAK-STATsignaling originating in larval muscle tissue is essential for theencapsulation of wasp eggs (Yang et al., 2015).Drosophila indirectflight muscles (IFMs) also act as an immune-responsive tissueessential for surviving bacterial challenges during adulthood(Chatterjee et al., 2016). During an infection, AMP productionoccurs in the adult IFMs. Importantly, reduction of AMPs throughknocking down Toll or Imd pathway components, or throughcompromising IFM structural integrity, limits an individual’s survival.Similarly, immune responsiveness through TLR-mediated signalingwas observed in zebrafish muscles following infection (Chatterjeeet al., 2016). These experiments establish muscle as a key immune-responsive tissue in the defense against infection in both invertebratesand vertebrates. Further supporting a shared connection betweenmuscle maintenance and innate immunity, we recently identifiedfondue (fon) as essential for maintaining the extracellular matrix(ECM) of the larval muscle attachment site (MAS) (Green et al.,2016). The common suite of secreted hemolymph proteins thataccumulate and function at the MAS were also found to act with Fonin its previously described role in coagulation (Bajzek et al., 2012;Lindgren et al., 2008; Scherfer et al., 2006).

The invasion of immune cells and inflammation followingmuscle injury is a standard strategy for cellular repair in vertebrates(Tidball and Villalta, 2010). However, individuals with myopathiesand muscular dystrophies often experience persistent immuneresponses in damaged muscle, which may contribute to diseaseprogression (Madaro and Bouché, 2014; Nitahara-Kasahara et al.,2016; Rosenberg et al., 2015; Villalta et al., 2015). Gene expressionprofiles obtained from Drosophila mutants with hypercontraction-induced myopathy (Montana and Littleton, 2006) and humanmuscular dystrophy patients (Chien et al., 1991; Haslett et al., 2002;Hathout et al., 2014) reveal an upregulation of genes involved inactin-dependent remodeling and chaperone transcripts, as well as adownregulation of metabolic and mitochondrial genes characteristicof metabolic stress in dystrophic muscle. Additional increases inclasses of immune-responsive genes upon muscle damage suggeststhat the capacity for immune responses to act not only in response to,but to potentially drive tissue damage, emphasizes the necessity forunderstanding the fundamental mechanisms regulating immune andtissue physiology.

Here, we have uncovered a tissue communication networklinking muscle maintenance and innate immune signaling. WhileReceived 27 March 2018; Accepted 15 November 2018

Department of Biochemistry and Molecular Biophysics, Kansas State University,Manhattan, KS 66506, USA.

*Author for correspondence ([email protected])

E.R.G., 0000-0002-1450-7166

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characterizing the muscle phenotypes of fon mutants (Green et al.,2016), we noted several immune responses resulting from damagedmuscle tissue, including the deposition of melanin at MASs,hemocyte recruitment to detached and torn muscles, and theactivation of both JAK-STAT and Toll signaling. Importantly, wealso show that JAK-STAT and Toll have the ability to establish areciprocal signaling network between tissues that sense and respondto cellular stress. Therefore, we conclude that muscle tissuemaintenance is coordinated with innate immunity in a multi-organresponse mediated through local JAK-STAT signaling and systemicToll activation.

RESULTSJAK-STAT signaling occurs in response to specific classesof muscle stressThe role of JAK-STAT signaling in tissue stress has been welldocumented (Myllymäki and Rämet, 2014). More recently, a noveland essential role for JAK-STAT activity within muscle tissue hasbeen described in the Drosophila response to parasitoid waspinfections (Yang and Hultmark, 2016; Yang et al., 2015).Furthermore, JAK-STAT signaling is upregulated at the bordersof epithelial wound sites and in the closely associated muscle layerof third instar (L3) larvae (Lee et al., 2017). Based on theseobservations, we wanted to determine whether JAK-STATsignaling could act as a local response to muscle tissue stressusing the JAK-STAT reporter, 10xSTAT92E-GFP (STAT-GFP).Bach et al. (2007) genetically engineered flies containing a reporterconstruct consisting of STAT92E binding sites fused to GFP. WhenJAK-STAT signaling is active, the transcription factor STAT92Etranslocates into the nucleus. With STAT in the nucleus, thetranscription factor associates with the tandem STAT-binding sitespresent in the reporter construct and leads to the production of GFPthat can be seen in both the nucleus and cytoplasm of cells (Fig. 1A).We devised a reporter screen using RNAi stocks to determine

which classes of muscle stresses could activate JAK-STAT inmuscle tissue. In healthy muscle, JAK-STAT signaling is held atlow levels (Fig. 1B,B′,H). Using the STAT-GFP reporter incombination with the muscle-specific mef2-GAL4 driver, wecould selectively knock down genes known to induce tissuedamage and assay for increased JAK-STAT activation in muscles.We identified representative genes where knockdown resultedin well-defined muscle phenotypes including degeneration,detachment, oxidative stress and mitochondrial stress to assess theability of each type of tissue stress to activate JAK-STAT.One of the most damaging phenotypes to tissue is muscle

detachment, which can occur as either a complete detachment ofmuscle from the ECM or a partial detachment compromising theconnection of the actin cytoskeleton to molecules in the extracellularspace. The knockdown of Tiggrin (Tig), a component of the larvalECM, causes muscles to round up and detach at indirect MASs(Bunch et al., 1998). The Drosophila gene steamer duck (stck)encodes PINCH, which is an adaptor protein associated with integrincomplexes whose loss leads to a detachment of the internal actincytoskeleton (Clark et al., 2003). RNAi knockdown of either Tig orstck results in muscle morphology defects and increased STAT-GFPexpression (Fig. 1C-D′,I,J). Based on these observations, it is notsurprising that muscles intentionally torn with forceps during livelarval dissections quickly induce a local JAK-STAT response incomparison to wild-type larvae that have been heat-killed prior todissection (Fig. S1). We also examined the effect of an external pinchwound, which preserves cuticle integrity while damaging theunderlying epithelial and muscle layers (Burra et al., 2013). Larvae

with pinch wounds show only a muted response to subtle muscledamage (Fig. S1), which may reflect the small degree of damageinflicted by this technique.

Skeletal muscle aging and several pathological conditions areassociated with a loss of metabolic homeostasis from mitochondrialdysfunction and increasing levels of reactive oxygen species (ROS)(Fabio et al., 2013). Tissue balance of ROS is maintained by a seriesof enzymes including Superoxide dismutase (Sod) and Catalase(Cat). Both overexpression and knockdown of Sod1 have beenreported to increase levels of ROS (Buettner et al., 2006; Cabreiroet al., 2011). Disruption of ROS balance through the reduction(Fig. 1E,E′,K) or overexpression (Fig. S1) of the Sod1 enzymeresults in active JAK-STAT signaling. Similarly, altering levels ofCat causes increased STAT-GFP in larval musculature (Fig. S1).Inducing mitochondrial stress through knockdown of mitochondrialtranscription factor a (TFAM) (Fig. 1F,F′,L) or parkin ( park)(Fig. S1) yielded the lowest levels of JAK-STAT activation from thechosen classes to screen (Greene et al., 2003; Larsson et al., 1998).Surprisingly, the myofibrillar unbundling phenotype present in thin(tn) RNAi fillets is not capable of activating JAK-STAT signaling(Fig. 1G,G′,M), suggesting that activation of this pathway is subjectto different cellular or molecular stresses.

JAK-STAT signaling is activated locally in response tomuscle damageOur results thus far show that knockdown of two genes required formuscle attachment (Tig and stck) activate local JAK-STATsignaling in muscle. We previously identified Fon as a novelECM organizer in preserving larval MASs (Green et al., 2016).Owing to its strong detachment phenotype and the ability to bypassthe embryonic lethality that complicates the analysis of similarmutants (i.e. integrin subunits), we next examined the muscle-specific activation of the JAK-STAT pathway in fon knockoutmutants. Indeed, removal of fon promotes widespread muscledetachment and elicits dramatic increases compared with wild typein STAT-GFP reporter expression in both the cytoplasm and nucleusof muscles (Fig. 2 A,A′,F,C,C′,H). This exceeds the increasedexpression of STAT-GFP that occurs in response to activation of thetemperature-sensitive, constitutively active JAK allele, hopTum-l

(Fig. 2B,B′,G). We noted in our reporter screen that basal levels ofJAK-STAT signaling are present in muscle, cuticle and epithelialcells in wild-type larvae, although there was a clear increase inmuscle-based JAK-STAT signaling from knockout of genes in themuscle detachment class (Fig. 1; Fig. S2). The majority ofhomozygous fon mutants (∼80%) share this expression pattern,although there were occasional instances (∼20%) where STAT-GFPin the epithelial layer underneath muscles appeared to experienceactivated JAK-STAT signaling as well (Fig. S2).

Genetic mutations characterized in Caenorhabditis elegans andDrosophila show that hypercontraction can act as a precursor tomuscle detachment (Bessou et al., 1998; Grisoni et al., 2002; Myerset al., 1996; Nongthomba et al., 2007; Nongthomba et al., 2004;Raghavan et al., 2000). Furthermore, expression profiling of thedominant MhcS1 allele in Drosophila adults revealed that immunesignaling was upregulated in response to hypercontraction-inducedmyopathy (Montana and Littleton, 2006). We wanted to examinewhether immune activation occurred at steps preceding muscledetachment (i.e. prolonged hypercontraction) and confirm thatimmune activation was responsive to muscle damage in larvalstages. We tested two temperature-sensitive hypercontractilemutants, MhcS1 and BrkdJ29, which contain hypercontractedmuscle as larvae and exhibit indented thorax phenotypes in adults

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as IFMs degenerate (Montana and Littleton, 2004). At restrictivetemperatures, bothMhcS1 and BrkdJ29 exhibit hypercontraction andactive JAK-STAT signaling (Fig. 2D–E′,I,J), suggesting that JAK-STAT acts as a local mediator in the progression towards muscledetachment.

Muscle stress induces a broad suite of immune responsesDuring our analysis of fon-induced muscle attachment, we noted theappearance of several phenotypes attributed to innate immunity. Inwild-type larvae, unchallenged individuals are free of melanization(Fig. 3A) (Binggeli et al., 2014; Tang, 2009; Tang et al., 2006). Itwas known that mutations in fon result in diffuse melanization atwound sites as well as the presence of melanotic tumors throughout

the hemocoel (Fig. S3) (Scherfer et al., 2006). A small percentage offon mutants (∼5%) present a unique melanization phenotype wheremelanin spontaneously accumulates at sites of muscle attachment(Fig. 3D; Fig. S3).

We utilized two methods to increase the frequency ofmelanization in fon mutants for further study: 1) uniform crushwounding to exacerbate tissue stress; and 2) dissection of larvaein excess L-DOPA substrate to increase phenoloxidase (PO)enzymatic activity (see Materials and Methods for details). Auniform crush-based trauma was administered to fon mutant larvaethrough gently compressing the dorsal surface along the body axis.This additional stress resulted in melanization of the dorsal vessel,particularly the pericardial cells, and occasionally localized melanin

Fig. 1. JAK-STAT is activated by specific classes ofmuscle stress. (A) Schematic of the JAK-STAT reporter, 10xSTAT92E-GFP (STAT-GFP), which containsSTAT binding sites enabling GFP transcription upon STAT activity in the nucleus. Activation of JAK-STAT signaling causes increased expression of theSTAT-GFP reporter in both the cytoplasm and nucleus. (B–G) Muscle phenotypes (F-actin, red) and nuclear positioning (DAPI, blue) in select RNAi knockdownsvia muscle-specific GAL4/UAS inducing different classes of cellular stress. (B′–G′) STAT expression in L3 larval muscles as measured with the STAT-GFPreporter (GFP, green). (B,B′) STAT levels are low or nearly undetectable in the cytoplasm and nucleus of unstressed larval muscles. (C,C′) RNAi knockdown ofTig in muscles leads to weakened tendon cell anchoring, but muscle attachments are maintained across hemisegments. Partial detachment is associatedwith cytoplasmic and nuclear increases in STATexpression (arrowheads). (D,D′) RNAi knockdown of stck, which encodes the integrin-associated PINCH protein,enhances STAT-GFP expression in muscles. Although knockdown of stck can cause muscle detachment, an attached muscle was selected for analysis tosimplify interpretation. (E,E′) Perturbations to oxidative stress induced through knockdown of Sod1 causes subtle disruptions to the sarcomeric spacing ofmuscles (see regions marked by yellow line in E) and STAT-GFP levels to weakly increase. (F,F′) Mitochondrial stress caused through knockdown of themitochondrial protein, TFAM, causes similar disruptions to muscle sarcomeres (yellow line in F) and activates JAK-STAT signaling. (G,G′) Myofibrillar unbundlingand muscle degeneration resulting from tn knockdown is not capable of activating JAK-STAT signaling. (H–M) Line plot analysis of STAT-GFP fluorescenceintensity throughout the cytoplasm and nucleus (white lines) in panels B′–G′. Green dots on each graph correspond to arrowheads marking nuclei in each imagepanel. GFP intensity from STAT-GFP control muscle in panel H is overlaid on line plots as a gray-filled profile in panels I–M. Additional examples of stresses canbe found in Fig. S1. Scale bars: 100 µm.

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accumulation at MASs (Fig. S3). Next, fillets were dissected andincubated in the presence of the PO substrate L-DOPA. In wild-type fillets free of muscle damage, melanin does not accumulate atMASs but may be found along dissection sites and within thecuticle (Fig. 3B,C). However, fon mutant fillets in the presence ofL-DOPA accumulate melanin that phenocopies spontaneously-occurring patterns at nearly 100% of MASs (Fig. 3D–F; Fig. S3).If tissue-based melanization is a result of muscle damage and notsimply a consequence of fon removal, we would expect to seemelanin accumulating in wild-type muscles intentionally damagedduring dissection or by trauma. Indeed, when muscles aredamaged during dissection and incubated in L-DOPA, melaninstrongly accumulates along the borders of damage sites (Fig. S3).Additionally, melanin accumulation is obvious at MASs upontear-induced muscle detachment and at the site of forcepspinching in conjunction with damage to the underlying musclehemisegments (Fig. S3). These data, taken together, show thatmuscle damage, whether through genetic manipulation or methodsof artificial muscle stress, is sufficient to trigger melanization-induced immune responses.Because melanization requires the activity of PO, which is

expressed only in a subset of hemocytes known as crystal cells, wewanted to determine whether hemocytes were localized to damagedmuscle tissue. During a wounding event, hemocytes are recruited tothe sites of damage to aid in repair through release of the PO enzymeand other secreted proteins (Krautz et al., 2014). In L3 larvae,hemocytes can be circulating throughout the hemolymph, reside assessile populations along the dorsal vessel, or in hematopoieticpockets between body wall muscles and epithelia (Holz et al., 2003;

Makhijani et al., 2011; Márkus et al., 2005; Stofanko et al., 2008;Zaidman-Rémy et al., 2012). Hemocytes are dispersed in thesepatterns along muscle hemisegments in unwounded larvae, but donot specifically localize to MASs (Fig. 3G; Fig. S3). In wild-typefillets with mechanical damage to muscles, hemocytes are recruitedto sites of tissue damage (Fig. 3H; Fig. S3). Similarly, hemocytesare targeted to detached muscles and attachment sites wheremuscles have begun to pull away from one another upon genetic lossof fon (Fig. 3I; Fig. S3). Taken together, these data indicate that lossof muscle maintenance is linked to the initiation of stress-basedsignaling and cellular immune activities.

Muscle damage caused by loss of fon activates pathogen-independent, systemic Toll signalingIn previous studies that examined the role of Fon in immunity, fonmutants constitutively expressed the AMP Drosomycin (Drs), andfon itself was identified as a target of Toll signaling (Scherfer et al.,2006). The muscle morphology of dissected wild-type larval filletsfeatures repeating hemisegments of broad, rectangular musclesstably anchored at MASs (Fig. 4B). In fonmutants, unstable muscleattachments lead to muscle detachment that generates extensivetissue damage (Fig. 4D). In the absence of pathogens, musclemorphology is maintained in wild-type fillets while still beingdisrupted in fon mutants (Fig. 4C,E).

We utilized the subcellular localization of the NF-κBtranscription factor, Dl, to evaluate Toll pathway activation uponloss of fon. In wild-type larvae without infection or damage, Tollsignaling is inactive and Dl is localized throughout the cytoplasm ofthe cell (Fig. 4A–B′). Upon Toll activation, Dl translocates into the

Fig. 2. JAK-STAT signaling is a local response to muscle damage via detachment. (A–E′) Expression of STAT-GFP reporter (green) in L3 larval musclestained with phalloidin (F-actin, red) and DAPI (blue). (A,A′) In normal muscle, STAT-GFP expression is at low levels. (B,B′) Activation of JAK-STAT signalingusing the constitutively active JAK allele, hopTum-l, increases STAT-GFP levels in both the cytoplasm and nucleus (arrowheads). (C,C′) Loss of fon causesdramatic increases in STAT-GFP both in the cytoplasm and the nucleus. For clarity and consistency, a muscle that remained attached was imaged. Lowermagnification images of STAT-GFP expression in fon mutants can be found in Fig. S2. (D–E′) In the hypercontractile mutants, MhcS1 and BrkdJ29, STAT-GFPexpression is increased throughout muscle tissue and concentrates in the nucleus (arrowheads). (F–J) Line plot analysis of STAT-GFP fluorescenceintensity along white lines drawn through muscles in panels A′–E′. Green dots on each graph correspond to arrows marking nuclei in each image panel. GFPintensity from STAT-GFP control muscle in panel F is overlaid on line plots as a gray-filled profile in panels G–J. Scale bars: 100 µm.

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fat body nucleus to initiate transcription of Toll-responsive genes(Fig. 4A). Dl staining is concentrated in the nuclei of fon nullmutants, indicating that a loss of Fon activates Toll signaling(Fig. 4D′). We also observe activation of Toll in the fat bodies ofwild-type larvae several hours after the muscle and underlyingepithelium is damaged via pinch wounding (Fig. S3). This nuclearlocalization of Dl is absent in muscle tissue of both wild-type andfon mutant larvae, narrowing Toll activation to a systemic ratherthan local response (Fig. S4). Previously, Drs expression inunchallenged fon mutants was detected using a Drs–GFP reporter(Scherfer et al., 2006). Complementary to these studies and ourobservations that fon mutants activate Toll in the fat body, Drstranscripts are dramatically increased in isolated fat bodies lackingfon (Fig. 4F).To determine whether microbes are important for Toll activation

in fon mutants, these same experiments were performed under

axenic, or germ-free, conditions. The appearance of muscledetachment and the translocation of Dl into the nucleus indicatethat the consequences of loss of fon are pathogen-independent(Fig. 4D–E′). We also tested whether the other major immuneresponse pathway, Imd, and its NF-κB transcription factor, Rel,were activated in response to fon-mediated muscle damage. Similarto Dl, Rel is restricted to the cytoplasm when Imd signaling isinactive and then translocates into the nucleus upon signaltransduction to begin immune-related gene expression (Stövenet al., 2000). We found that Rel is retained in the cytoplasm of wild-type and fon mutant fat bodies and transcription of the AMPdiptericin (dpt) is not upregulated in response to muscle stress(Fig. S4). Therefore, Toll signaling is activated systemically andselectively in response to muscle damage owing to loss of fon.

fon interacts with genes necessary for the activation of TollsignalingBecause knockdown of Fon induces systemic Toll activation, wewanted to determine whether canonical components of Tollsignaling are required. We implemented a fon-sensitizedbackground ( fonΔ24/+; da-GAL4) screen using the GAL4/UASsystem to target genes at regulatory points in the Toll signaltransduction cascade (Brand and Perrimon, 1993). In both wild-typelarvae and fon heterozygotes, rarely is muscle detachment observed(Fig. 5A,E,I). However, when crossed to lines with candidate genesthat interact with fon, genetic interactions would lead to anenhancement of muscle detachment greater than that observed inlines where the candidate gene alone was knocked down oroverexpressed. Candidate lines were chosen to simulate the geneticactivation of Toll signaling, i.e., either the overexpression of genes(UAS-SPE, UAS-dl, US-Dif, UAS-Toll10B) at key activation stepsor the knockdown of inhibitors (UAS-cact RNAi) present in thepathway.

We first tested the effects of knocking down cactus (cact), aselongated pupal phenotypes present in cact mutants are similar tothose observed in fon mutants (Green et al., 2016; Letsou et al.,1991). The NF-κB inhibitor, cact, is responsible for sequesteringthe transcription factors, Dif and Dl, in the cytoplasm until Tollsignal transduction proceeds. When cact is ubiquitously knockeddown using RNAi, muscles remain intact (Fig. 5B,I). Knockingdown cact in the fon-sensitized background causes dramatic,widespread muscle detachment denoting a genetic interactionbetween fon and cact (Fig. 5F,I). We note that only smalldecreases in cact transcripts (Fig. 5J) are necessary to inducemuscle detachment as knockdown of cact at 29°C in the fon-sensitized background results in lethality. As verification that thiseffect was due to knockdown of cact transcripts and not off-targeteffects, we observed the expected melanization and Toll activationphenotypes upon tissue-specific RNAi knockdown (Fig. S5)(Lemaitre et al., 1995; Qiu et al., 1998). Overexpression of Dl inthe fon-sensitized background results in low levels of muscledetachment, although this trend is not statistically significant(Fig. 5G,I). Dif and Dl have been shown to play redundant rolesin larval immune tissues that could account for the minimal effectexerted by overexpression of Dl (Lemaitre et al., 1995; Rutschmannet al., 2000). However, genetic interactions between fon and twoimportant pathway members, Dif and Tl, could not be determineddue to lethality of these crosses at temperatures as low as 18°C(Fig. 5I).

We also investigated genes involved in upstream activationevents that take place in the extracellular space for ligand-receptorbinding. Spatzle-processing enzyme (SPE) is a protease required for

Fig. 3. Loss of Fon activates innate immune processes. (A–F) Presence ofmelanin in wild-type (WT) versus fonΔ17 homozygous mutants visualized inexterior views (A,D) and dissected muscle fillets (B,C,E,F). (A) Wild-typelarvae lack a visible melanization response throughout the body cavity orMASs (black arrowheads). (B) Addition of the phenoloxidase substrate,L-DOPA, allows for conversion into melanin which collects non-specificallythroughout the cuticle of wild-type larvae. Areas containing MASs along thehemisegmental borders are marked with yellow dashed boxes. (C) Wild-typemuscle attachments (black arrowheads) imaged at higher magnification arefree of melanin. (D) Melanin is spontaneously deposited at MASs (blackarrowheads) in low percentages (∼5%) of fonΔ17 mutant larvae.(E) Melanization at MASs (yellow boxes) can be induced by providing excessL-DOPA substrate to dissected fillets. (F) High magnification of melanindeposits at muscle attachments of fonΔ17 larvae observed upon additionof L-DOPA (black arrowheads). (G–I) Distribution of hemocytes at MASs infilleted larvae. Low magnification images can be found in Fig. S3.(G) Hemocytes (detected through labeling for Hemese) are found at low levelsnear intact MASs. (H,I) In wild-type muscles that have been mechanicallydamaged during dissection (H) or upon fon-mediated muscle detachment (I),hemocytes are recruited to sites of muscle attachment and/or damagedmuscles. Arrowheads denote the MAS in panels G–I. Scale bars: 1 mm in A,D;500 µm in B,E; 100 µm in C,F–I.

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cleavage of the proenzyme, pro-Spatzle, into its active form (Spz),which binds to Toll to initiate signal transduction (Mulinari et al.,2006). Overexpression of SPE alone is not sufficient to causemuscle detachment (Fig. 5D,I). However, SPE overexpression in aheterozygous fon background disrupts muscle detachment(Fig. 5H,I). These results show that fon functions with membersof the Toll pathway to mitigate muscle damage via detachment.The major output of Toll signaling is the expression of AMPs,

which eliminate pathogens through mechanisms that are presentlynot understood (Lemaitre and Hoffmann, 2007). AMPoverexpression has recently been shown to be a driving force inDrosophila neurodegeneration (Cao et al., 2013). We reasoned thata potential mechanism for disrupting muscle tissue could comefrom the constitutive expression of AMPs as a result of systemicToll activation. Using the same sensitized background approach, weexamined the effects of overexpressing specific AMPs on musclearchitecture. We tested a subset of AMPs including Drs,Metchnikowin (Metch, also known as Mtk), and Drosocin(Dro), selected to represent expression that encompasses Tolland/or Imd signaling. Although the ubiquitous overexpression ofthese AMPs did not significantly enhance muscle detachment,we did see the appearance of hypercontracted regions within themuscle when AMPs were overexpressed in a fon-sensitizedbackground (Fig. S6). We then overexpressed two of these AMPsin either neural (C155-GAL4) or muscle (mef2-GAL4) tissue duringlarval stages. This tissue-specific overexpression of AMPs wasnot sufficient to significantly cause muscle hypercontraction,although there were populations of analyzed muscle fillets thatcontained large amounts of hypercontracted muscles (Fig. S6).Although these data suggest that AMPs could contribute to muscledefects, overexpression of AMPs alone is not sufficient to drivemuscle damage.

JAK-STAT and Toll signaling are coordinated during musclestressHaving identified a breadth of immune responses at both the localand systemic level, we wanted to understand how muscle and fatbody cells could communicate to coordinate an efficient overallresponse to muscle stress. To determine the potential for a reciprocalnetwork between fat body and muscle cells, we chose to ectopicallyactivate JAK-STAT signaling using genetic tools. Constitutively

active forms of JAK (hopTum-l) or the Tl receptor (Tl10B and Tl3) areavailable as both dominant alleles and UAS constructs forubiquitous or tissue-specific expression, respectively. Usingpreviously described assays to report JAK-STAT (STAT-GFP) orToll (nuclear Dl translocation) activation, we induced each of thesepathways in healthy tissue and examined either fat body or muscletissue to determine mechanisms of tissue crosstalk (Fig. 6A).

In hopTum-l larvae,muscles appear similar towild-type (Fig. 6B,D).When JAK-STAT signaling is activated in all tissues, we observe Dlredistributing from the cytoplasm as seen in wild-type fat bodies(Fig. 6C) into the nucleus coincident with active Toll signaling(Fig. 6E). Next, we wanted to determine whether tissue-specificactivation of JAK-STAT could induce fat body-based Toll signaling.Activation of JAK-STAT in fat body, muscle, tendon or hemocytesalone was not sufficient to induce activation of systemic Tollsignaling (Fig. S7). Conversely, when Toll signaling is constitutivelyactivated in all larval tissues with the Tl3 allele, STAT-GFP expressionincreases in damaged muscle in comparison to undamaged muscleand is similar to ubiquitous hopTum-l activation (Fig. 6F–H′,K–M).This effect persists when UAS-Tl10B is expressed using two fat bodydrivers (Fig. 6I–J′,N,O). These data suggest that JAK-STAT and Tollfunction in a signaling network involvingmultiple tissues in responseto muscle stress and damage.

DISCUSSIONWe have made the unanticipated discovery that innate immuneactivation occurs upon muscle stress at both the local and systemiclevels. First, we observe increases in JAK-STAT signaling upon lossof muscle homeostasis, which has been described in many tissuestresses to act as a local mediator of immune induction and geneexpression (Myllymäki and Rämet, 2014; Srinivasan et al., 2016).Our data also shows that specific muscle stresses activate JAK-STAT signaling (Figs 1, 2), further emphasizing that immunepathways are responsive to the physiological states of tissues. Weshow that muscle detachment caused by loss of fon specificallyactivates Toll signaling and that this signaling is restricted to the fatbody (Fig. 4; Fig. S4). Moreover, our experiments indicate thatJAK-STAT signaling is capable of activating Toll in the fat body todrive the systemic immune response and vice versa (Fig. 6). Wehave compiled these data into a proposed model (Fig. 7) thatoutlines how JAK-STAT signaling and Toll signaling are used to

Fig. 4. Toll signaling is activated in fonmutants. (A) Schematic showing Dl localization during Toll signaling. Dl is primarily cytoplasmic when Toll signaling isturned off in L3 fat body cells. Following Toll activation, Dl moves into the nucleus to induce gene expression. (B–E) Muscle fillets of dissected L3 wild-type andfonΔ17 mutants raised in either normal or axenic conditions and visualized with phalloidin (F-actin, red). (B′–E′) Dl localization (green) in larval fat bodytissue. A single fat body cell is outlined in white for each panel. (B,C)Wild-type muscles are rectangular and firmly anchored to adjacent muscles and tendon cellsin both the presence and absence of pathogens (axenic). (B′,C′) Dl is localized to the cytoplasm of fat body cells regardless of the presence or absence ofpathogens. (D,E) Muscles round up and detach (arrowheads) upon loss of fon, independent of the presence of pathogens. (D′,E′) Dl is enriched in thenucleus of fonmutant fat body cells in both normal and axenic conditions. (F) Mean±s.e.m relative transcript levels ofDrsRNA collected from the pooled fat bodiesof wild-type larvae, larvae expressing fon alleles (Δ17/Df and Δ24/Df ), and larvae demonstrating fat-body induced Toll (Tl) overexpression, Cg>Tl10B. Scale bars:500 µm in B–E; 50 µm in B′–E′.

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sense and respond to damage in a complex network involving tissuecoordination.Muscle damage results in a broad array of humoral and cellular

events, including the recruitment of hemocytes, which are known tosecrete a variety of bioactive molecules at wound sites (Krautz et al.,2014). A subset of hemocytes called crystal cells are responsible forsecreting PO to activate melanization, a critical step in hardeningand stabilizing the clot when melanin polymerizes around the softclot structure. Hemocyte localization to damaged MASs and thestrengthening role of melanization provides solid rationale for theinvolvement of hemocytes in a stabilizing response at damagedMASs (Fig. 7). Mutations in genes important for hemocyte functionor misregulation of PO activity result in the formation of melanotictumors (Minakhina and Steward, 2006). Interestingly, constitutivelyactive mutants of both Tl and hopscotch (hop) produce theaccumulation of melanotic tumors in Drosophila larvae andadults, exposing relationships between the melanotic cascade andtwo major immune signaling pathways, Toll and JAK-STAT(Harrison et al., 1995; Lemaitre et al., 1995). Furthermore, Tollsignaling is required for both hemocyte recruitment andmelanization (Schmid et al., 2014). These observations suggest an

intricate orchestration of the cellular and humoral systems during theimmune response, the details of which remain to be elucidated.

Interesting future directions suggested by our model includedissecting the extracellular events that respond to damage-basedsignals, identifying secreted ligands, and determining thecontribution of hemocytes that link JAK-STAT and Toll signalingfollowing muscle damage. It has previously been shown that theToll ligand Spz is produced and secreted primarily from hemocytes(Irving et al., 2005). Upd ligands (Upd1–Upd3) necessary for JAK-STAT activation are also secreted by hemocytes in specificcircumstances (Yang and Hultmark, 2016; Yang et al., 2015),making these mobile cells a prime candidate for sensing andresponding to both muscle and fat body cues through ligandexpression (Fig. 7). Skeletal muscle has also been shown to produceUpd ligands during homeostatic communication and may act as asource for long-range secretion (Zhao and Karpac, 2017). Atpresent, our efforts are concentrated on understanding how multipletissues function in concert to modulate signaling events duringimmune responses induced by muscle damage.

In a normal infection, the dramatic expression of AMPs targetspathogens, eliminating and deactivating foreign molecules through a

Fig. 5. Genetic interactions between fon and Toll pathway components enhance muscle detachment. (A–H) Two hemisegments of muscle fillets stained withphalloidin (F-actin, green) in a wild-type control, candidate RNAi lines alone (B–D) or in the fon-sensitized genetic background, fonΔ24/+; da-GAL4 (E–H). (A,E)Morphological defects are absent in the muscles of wild-type and fonΔ24/+; da-GAL4 larval fillets. (B) RNAi knockdown of the NFκB inhibitor, cact, alone does notdisrupt muscle attachment. (C,D) Overexpression of Dl or SPE has no obvious consequences for muscle attachment stability. (E–H) In comparison to heterozygousfonΔ24/+ alone (E), loss of cact (F) or the overexpression of dl (G) or SPE (H) in a fon-sensitized background enhances muscle detachment (arrowheads).(I) Mean±s.e.m quantification of muscle detachment of select genotypes ( fonΔ24; da>yw, n=16; da>cact RNAi, n=12; fonΔ24; da>cact RNAi, n=21; da>UAS-SPE,n=20, fonΔ24; da>UAS-SPE, n=19; da>UAS-dl, n=10; fonΔ24; da>UAS-dl, n=13; da>UAS-spz(FL) #1, n=20; fonΔ24; da>UAS-spz(FL) #1, n=12); da>UAS-spz(FL) #2,n=17; fonΔ24; da>UAS-spz(FL) #2, n=10; da>UAS-Dif, n=16). Lethality of UAS-Dif and UAS-Toll10B combinations at and above 18°C prevented a similar larvalanalysis. Dots in each plot indicate results for individual larval fillet samples. (J) Mean±s.e.m effectiveness of RNAi knockdown of cact transcripts through cactRNAi #3 determined using qPCR. RNAi phenotypes of additional cact RNAi lines tested can be found in Fig. S5. P-values determined via Kruskal–Wallisstatistical test; ***P

variety of destructive mechanisms. When AMPs lack explicitpathogenic targets, the action of AMPs on healthy tissue creates aparadox for innate immune activation during sterile tissue damage(Cao et al., 2013). In the context of muscle damage, AMPsmay act onstressed muscle tissue to exacerbate damage, which we see in theappearance of hypercontraction as a result of AMP overexpression(Fig. S6). InDrosophila, excessive levels of AMPs have already beenshown to induce neurodegeneration when activated through neuralbacterial infections orartificial neuronal-specific expression (Cao et al.,2013). The fact that several key regulatory points in Toll signaling, andpossibly the activity of AMPs, intensify muscle detachment in a fon-sensitized background seems to suggest that prolonged periods ofsystemic immune signaling can act as an additional source of cellularstress in already damaged tissue (Damage 3 in Fig. 7).It is consistent that broad-scale injuries induced during live

dissection or caused by intentional wounding produced a robustincrease in STAT-GFP expression similar to muscle detachment(Fig. 1; Fig. S1). Although a less invasive and aggressive form ofwounding via an external pinch wound produced only low levels ofJAK-STAT signaling or hemocyte recruitment (Fig. S3), this type of

injury sufficiently activated Toll signaling (Fig. S3), which suggestsdifferential mechanisms for Toll activation. This is supported bywork demonstrating activation of the larval humoral responsefollowing a gentler method of pinch wounding, which was shown tobe independent of hemocytes (Kenmoku et al., 2017). It should benoted that the overall robustness of immune responses followingmuscle stress are attenuated in comparison to those observed duringinfection. One explanation for this difference could be the urgencyrequired to eradicate invading pathogens, which is not as imperativein sterile tissue damage.

It was surprising that muscle detachment elicited such a strongincrease in JAK-STAT signaling, as muscle degeneration had nodetectable effect on reporter expression (Fig. 1). Distinct differencesin the pathology underlying each type of muscle stress or damagemay be related to the degree towhich tissue integrity is disrupted and/or the nature of molecules released into the extracellular environment.There is evidence that the manner of cell death (necrosis or apoptosis)releases different subsets of cellular signals (Kolb et al., 2017). Subtledetails in these differences may help to explain why programmed celldeath fails to activate immunity during development, while injuries

Fig. 6. JAK-STAT and Toll pathways are activated in a reciprocal signaling network. (A) Schematic showing experimental design used to determine therelationship between Toll and JAK-STAT signaling. When Toll signaling is activated in the fat body, Dl staining concentrates in the nucleus. Activation of JAK-STATresults in increasedSTAT-GFPexpression in both the cytoplasmand nucleus ofmuscles. (B–E) Impact of JAK-STAT signaling in L3muscle on systemic Toll signaling.(B) Two hemisegments of wild-type muscle (F-actin, red) with stable attachment sites. (C) The NFκB transcription factor, Dl (Dorsal, green), localizes to thecytoplasm of fat body cells (outline, inset). Nuclei are stained with DAPI (blue). (D) Constitutively active hopTum-l mutants have muscles with no visible defects.(E) Dl translocates into the nucleus of fat body cells following JAK-STAT activation via the hopTum-l mutation (outline, inset). For tissue-specific expression of hopTum-l

see Fig. S6. (F–J′) Analysis of JAK-STAT signaling following Toll activation. (F,F′) In wild-type muscles, expression levels of the STAT reporter STAT-GFP (green)are low. (G,G′) Overall activation of the Drosophila JAK allele, hopTum-l, causes STAT-GFP levels to increase in both the cytoplasm and nuclei (arrowheads) ofmuscle tissue. (H–J′) Constitutively active Toll signaling using the activated Toll allele Tl3 (H,H′) or UAS-Tl10B expressed using a fat body- and salivary gland‐specificdriver (I,I′) or fat body- and hemocyte-specific driver (J,J′) increases STAT levels in larval muscle. (K–O) Line plot analysis of STAT-GFP fluorescence intensitiesmeasured from white lines drawn in panels F′–J′. Green dots on each graph correspond to arrowheads marking nuclei in each image panel. GFP intensity fromSTAT-GFP control muscle in panel K is overlaid on line plots as a gray-filled profile in panels L–O. Scale bars: 500 µm in B,D; 50 µm in C,E; 100 µm in F–J′.

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and subsequent tissue stress later in life induce robust immuneresponses (Pradeu and Cooper, 2012).Models for initiating innate immune responses have been

proposed through either pathogen-associated molecular patterns(PAMPs) or damage-associated molecular patterns (DAMPs).Examples of ‘damage-based’ innate immune activation can be foundthroughout the invertebrate and vertebrate literature (for reviews seeChen and Nuñez, 2010; Kono et al., 2014; Shaukat et al., 2015). Weshow that both muscle detachment and systemic immune activationare pathogen-independent, which could implicate the release of aDAMP as part of this tissue network. More recently, efforts tounderstand DAMP-based immune activation have focused on theidentification of molecules capable of initiating immune signalingupon tissue damage. Examples of identified DAMPs includeintracellular components such as chromatin, nucleotides (ATP),ROS, cytoskeletal components (Mhc, actin), or fragments of theECM or basement membrane that are released and recognized asforeign to activate immune pathways, often through Toll-like receptor(TLR) signaling (Kono et al., 2014). Complementary studies inDrosophila and vertebrates showed that F-actin released during tissuedamage is sufficient to elicit an immune response (Ahrens et al., 2012;Srinivasan et al., 2016). Torn and leaky membranes characteristicof muscle damage in chronic diseases present opportunities forinvestigations that integrate DAMP release and the immunephenotypes noted in clinical descriptions. The large arsenal ofgenetic and molecular tools in Drosophila will continue to proveuseful in the identification of novel conserved DAMPs.Precedence for mechanical damage activating NF-κB pathways

was previously reported in the mdx mouse model of Duchennemuscular dystrophy (DMD) and in patients with a variety of musclediseases including DMD, skeletal muscle atrophy and cachexia-induced muscle wasting (Acharyya et al., 2007; Evans et al., 2009;Kumar and Boriek, 2003; Li et al., 2008; Messina et al., 2011;Monici et al., 2003; Mourkioti and Rosenthal, 2008; Peterson andGuttridge, 2008; Peterson et al., 2011). In addition to Toll acting asthe main signaling pathway in many types of infections ininvertebrates, mitigating vertebrate TLR signaling implicated inthe pathology of myositis and inflammatory myopathies is being

explored as a therapeutic intervention (Rayavarapu et al., 2013;Tournadre and Miossec, 2013). Drosophila has yielded manyinsights into vertebrate TLR immune response, which garnered aNobel Prize in 2011 (Lemaitre et al., 1996). Because of the largelyconserved nature of Toll signaling betweenDrosophila and humans(Buchon et al., 2014; Valanne et al., 2011), determining therelationship between tissue stress and Toll signaling could havebenefits in defining new methods of immune activation and inproviding novel perspectives on pathological conditions.

MATERIALS AND METHODSFly geneticsFlies were raised on standard cornmeal medium at 25°C unless otherwisespecified. The control stock used in all experiments was w1118. Two fon nullalleles, fonΔ17 and fonΔ24 (Bajzek et al., 2012) were used to remove fon andpaired with the deficiency stock,Df(2L)Exel6043. Other mutant alleles usedin experiments were BrkdJ29 (a gift from Troy Littleton, MassachusettsInstitute of Biology, Cambridge, MA, USA), hopTum-l (BloomingtonDrosophila Stock Center, BL-8492),MhcS1 (a gift from Troy Littleton), andTl3 (BL-3338). The following GAL4 lines were used to direct tissue-specificexpression: da-GAL4 (originally BL-37291 outcrossed ten times tow1118 toremove background lethals), C155-GAL4 (BL-458), Cg-GAL4 (BL-7011),mef2-GAL4 (BL-27390), sr-GAL4, UAS-CD8-GFP/TM6 (a gift fromTalila Volk, Weizmann Institute of Science, Rehovot, Israel), hml-GAL4,UAS-2xEGFP (BL-30140) and ppl-GAL4 (a gift from Len Dobens,University of Missouri-Kansas City, Kansas City, MO, USA). Stocksanalyzed in screens include UAS-cat (BL-24621), UAS-Dif (BL-22201),UAS-dl (BL-9319), UAS-Dro (a gift from David Wassarman, University ofWisconsin-Madison, Madison, WI, USA), UAS-Drs (a gift from DavidWassarman), UAS-hopTum-l (a gift from Michelle Starz-Gaiano, Universityof Maryland-Baltimore County, Baltimore, MD, USA), UAS-Metch (a giftfrom David Wassarman), UAS-Sod1 (BL-33605), UAS-SPE [a gift fromWon-Jae Lee; (Jang et al., 2006)], UAS-spz(FL) #1 (a gift from TonyIp, University of Massachusetts Medical School, Worcester, MA, USA),UAS-spz(FL) #2 (a gift from Tony Ip), UAS-Toll10B (BL-58987), UAS-cactRNAi #1 (BL-31713), UAS-cact RNAi #2 (BL-34775), UAS-cact RNAi #3(BL-37484), UAS-cat RNAi (BL-34020), UAS-Sod1 RNAi (BL-22491),UAS-stck RNAi (BL-31536), UAS-Tfam RNAi (BL-17644), UAS-Tig RNAi(BL-31570; RNAi validation in Green et al., 2016), UAS-tn RNAi(BL-31588; RNAi validation in Brooks et al., 2016), and UAS-park

Fig. 7. Model of damage-based tissue communication.Schematic representation of the tissue communication networkthat is activated following disruptions to muscle homeostasis.Muscle health depends on muscle integrity and the strongattachment of muscle (red) to tendon (green, asterisk) via ECMinteractions. Muscle hypercontraction (Damage 1) andweakened MASs that progress to detachment (Damage 2)generate stress responses that activate local and systemicimmune responses. Muscle damage prompts a series ofcellular immune responses including hemocyte recruitment andmelanization. Locally, JAK-STAT signaling is activated inmuscle tissue through the binding of Upd ligands to theDomeless (Dome) receptor to induce expression of immune-responsive genes. Toll signaling is activated in conjunction withthe JAK-STAT pathway in a reciprocal network. Upon Toll signaltransduction, Dl moves into the nuclei of fat body cells to activateToll-responsive genes such as the AMP, drosomycin. Otherforms of cellular stress in muscle tissue or possibly incoordinating tissues such as epithelium (Damage 3) maycoincide with destabilization of muscle attachment andcontribute to further loss of muscle maintenance.

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RNAi (BL-38333; RNAi validation in Brooks et al., 2016). The reporterstock used in these experiments was 10xSTAT92E-GFP (BL-26197).

Mutant alleles and genetic constructs were maintained over theappropriate balancer chromosome: FM7C (I), Cyo-Act-GFP or Cyo, Tb(II), or TM6, Tb (III). Individuals were chosen by selection against the Tb orGFP marker for second and third chromosome crosses or gender in firstchromosome balanced alleles. All temperature-dependent crosses wereperformed at 29°C with the following exceptions: (1) crosses involvingtemperature-sensitive alleles BrkdJ29 and MhcS1 were raised at 29°C andheat shocked for 1 h at 37°C immediately before dissection; (2) to bypassembryonic development, hopTum-l and UAS- hopTum-l crosses were shiftedfrom 18°C to the permissive temperature at 29°C following embryogenesis;(3) crosses for the muscle-specific RNAi knockdown of stck were shiftedfrom 18°C to 29°C at L1 to overcome lethality in embryogenesis; (4)experiments using cact RNAi #3 were performed at 25°C to avoid earlylethality in combination with the fon-sensitized background. Othercact RNAi stocks (#1 and #2) were analyzed at 29°C.

Immunostaining and microscopyWandering L3 larvae were dissected to either retain fat body tissue or isolatemuscle fillets, and the tissue fixed in 4% formaldehyde. All dissections wereperformed on larvae which were heat-killed, chilled and dissected in ice-coldPBS, except for larvaewhere the effects ofmechanical damage onmusclewereexamined or to test the effect of live dissection on STAT-GFP levels (Fig. S1).To induce mechanical damage to muscle tissue, larvae were dissected live inHL3 buffer and forceps were used to tear muscles along the middle of ahemisegment on both halves of the fillet, being careful to avoid damage toother tissues. Dissectionswere left unfixedwith all tissues intact for 15–30 minbefore resuming standard fixation and staining protocols. Extraneous tissueswere removed after fixation for the visualization of muscles. Crush-basedtrauma was performed by gently compressing and rolling L3 larvae betweentwo microscope slides on a level surface to create uniform stress to the bodycavity, pinch wounding was performed using forceps (Burra et al., 2013) withcare taken to preserve cuticle integrity but to encompass a large enough area toinduce damage to the muscle layer. Larvae were dissected 2 h after pinchwounding and 4–6 h post-wound to assess STAT-GFP activation.

Tissues were stained with the following primary antibodies: mouse anti-Dl[1:200; 7A4, Developmental Studies Hybridoma Bank (DSHB)], rabbit anti-Hemese (1:1000; a gift from Dan Hultmark, Umeå University, Umeå,Sweden), and mouse anti-Relish-C (1:400; 21F3, DSHB). Fluorescence wasdetected using the following secondary antibodies: Alexa Fluor anti-mouse 488 and Alexa Fluor anti-rabbit 488 (1:400; A-11001, A-11034,Molecular Probes). F-actin was labeled with phalloidin 488, 594 or 647(1:400; A12379, A22283, A22287, Molecular Probes). Images werecaptured using a Zeiss 700 confocal microscope. Image processing andanalysis was performed using a combination of Zen Black (Zeiss), ImageJ(NIH) and Adobe Photoshop.

qPCR analysisTranscript levels were assessed using quantitative PCR (qPCR) to verify RNAiknockdown and to compare gene expression amongst genotypes. Total RNAwas collected in triplicate from individual wandering L3 larvae using theRNeasy Mini Kit (Qiagen). Fat body-specific RNAwas obtained by isolatingthe fat bodies of five L3 larvae and homogenizing isolated tissues in ice-coldRLT buffer (Qiagen). Synthesis of cDNA from 125 ng RNA was performedusing the qScript XLT cDNA Supermix kit (Quantabio). Dilutions of cDNAwere optimized according to each primer set and combined with PowerUpSYBRGreen Master Mix (Thermo Fisher). The following primers and cDNAdilutions were used: rp49 forward 5′-GCCCAAGGGTATCGACAACA-3′,reverse 3′-GCGCTTGTTCGATCCGTAAC-5′ (1:50) (generated via FlyPrimerBank; Hu et al., 2013); Drs forward 5′-CCCTCTTCGCTGTCCTGA-3′, rev-erse 3′-GCGTCCCTCCTCCTTGC-5′ (1:50) (Deng et al., 2009); cact forward5′-CTCACTAGCCACTAGCGGTAA-3′, reverse 3′-CCCGAATCACTGGT-TTCGTTT-5′ (1:50) (Wang et al., 2015); and dpt forward 5′-ATTGGACTG-AATGGAGGATATGG-3′, reverse 3′-CGGAAATCTGTAGGTGTAGGT-5′(Chatterjee et al., 2016). All primers were synthesized at Integrated DNATechnologies (IDT). Quantitative transcript levels were obtained using the2-ΔΔCt method and graphed as mean±s.e.m. using GraphPad Prism 6.0.

Phenotypic quantification and statistical analysisDetachmentImages were quantified as described (Green et al., 2016). Muscles wereconsidered detached if they had rounded up following detachment or werebeginning to strip away from the attachment site. Percent detachment wascalculated by dividing the number of hemisegments containing one or moredetached muscles by the total number of hemisegments within the fillet.These percentages were compiled in GraphPad Prism 6.0 and graphicallyrepresented as a dot plot.

HypercontractionMuscles containing differentially compressed regions of sarcomeres werescored as ‘hypercontracted’ as previously defined (Montana and Littleton,2004; Montana and Littleton, 2006). Percent hypercontraction was calculatedin the samemanner as percent detachment, input intoGraphPad Prism 6.0 andgraphed as mean±s.d. dot plots.

Line plot intensitiesAll images within a particular analysis were taken using the same power andgain configurations. Fluorescence intensities of both STAT-GFP andphalloidin were analyzed along a single line across the length of ventrallongitudinal muscles using the plot profile feature in ImageJ. Line plots ofhypercontracted muscles were taken to illustrate the spacing of sarcomeresthat correspond to the peaks and valleys of phalloidin staining. For STAT-GFP expression comparisons, axis rangewas set to best fit the genotype plotwith the most signal intensity. Control plot profiles were traced and overlaidon experiment plots to highlight differences in plot intensities. Note thatcontrol STAT-GFP muscles in both Figs 1 and 2 contain two copies of theSTAT-GFP reporter as well as the fonΔ17 allele (Fig. 2; Fig. S2), which wasrecombined with the STAT-GFP reporter and analyzed as a homozygote.Larvaewith the following genetic manipulations contain a single copy of theSTAT-GFP reporter: RNAi lines (Fig. 1; Fig. S1), dominant alleles (Figs 2, 6),and overexpression constructs (Fig. 1; Figs S1, S6).

Statistical analysisStatistical analyses were performed in GraphPad Prism 6.0 using theKruskal–Wallis test to analyze non-Gaussian distributions of three or moreunmatched genotypes. Significance values are listed for each quantificationwithin the figure legend.

DOPA incubationLarvae were live dissected and washed in cold PBS. Muscle fillets were thenincubated in L-DOPA (Cayman Chemical) solution (60 mM dissolved inPBS) for 1 h at 25°C in the dark to allow melanization to proceed. Filletswere then washed and fixed in 4% formaldehyde and stained with phalloidin(1:400, Molecular Probes) to determine muscle morphology at points ofmelanization. Images were taken on a Nikon 80i and processed usingImageJ (NIH) and Adobe Photoshop.

Axenic conditionsAxenic larvae were generated using the protocol generated by Sabat et al.(Sabat et al., 2015). In a sterilized hood, embryos were dechorionated andsterilized using bleach and 70% ethanol solutions. Sterilized embryos weretransferred to autoclaved food vials until individuals matured to the larvalstage. Larvae were then collected and dissected as described above. Themicrobe status of axenic lines was analyzed by means of growing overnightcultures on Luria broth (LB) containing larval lysates from either normal oraxenic conditions. No bacterial growth was observed in axenic linescompared to an LB only control, whereas larval lysates not sterile and grownon normal food showed obvious bacterial growth.

AcknowledgementsWe thank Len Dobens, DanHultmark, Tony Ip,Won-Jae Lee, Troy Littleton, MichelleStarz-Gaiano, Talila Volk and David Wassarman for contributing reagents; DavidBrooks for reviewing of the manuscript; and Michael Kanost, Maureen Gorman andNeal Dittmer for valuable consultation on immunity experiments. We also thank theBloomington Drosophila Stock Center for stocks used in this study and theDevelopmental Studies Hybridoma Bank, created by the National Institute of Child

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Health and Human Development of the National Institutes of Health (NIH) andmaintained at the University of Iowa for monoclonal antibodies.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: N.G., E.R.G.; Methodology: N.G., E.R.G.; Validation: N.G.;Formal analysis: N.G.; Investigation: N.G., J.W., A.B., M.Z.; Resources: E.R.G.;Writing - original draft: N.G., E.R.G.; Writing - review & editing: N.G., E.R.G.;Visualization: N.G., E.R.G.; Supervision: E.R.G.; Project administration: E.R.G.;Funding acquisition: E.R.G.

FundingWe acknowledge funding from the National Science Foundation for a GK-12 awardto N.G. (0841414) and from the National Institutes of Health for RO1 (AR060788)and K-INBRE (GM103418) grants to E.R.G. Deposited in PMC for release after 12months.

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.217943.supplemental

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A tissue communication network coordinating innate immune ......hemocyte recruitment to detached and torn muscles, and the activation of both JAK-STAT and Toll signaling. Importantly,