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A drosomycin-GFP reporter transgene reveals a local immune response in Drosophila that is not dependent on the Toll pathway

1998; Springer Nature; Volume: 17; Issue: 5 Linguagem: Inglês

10.1093/emboj/17.5.1217

1460-2075

Dominique Ferrandon, Alain C. Jung, Marie‐Claire Criqui, Bruno Lemaître, S. Uttenweiler‐Joseph, Lydia Michaut, Jean‐Marc Reichhart, Jean‐Sébastien Hoffmann,

Antimicrobial Peptides and Activities

Article2 March 1998free access A drosomycin–GFP reporter transgene reveals a local immune response in Drosophila that is not dependent on the Toll pathway D. Ferrandon Corresponding Author D. Ferrandon Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du Centre National de la Recherche Scientifique, 15, rue René Descartes, F67084 Strasbourg, France Search for more papers by this author A.C. Jung Corresponding Author A.C. Jung Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du Centre National de la Recherche Scientifique, 15, rue René Descartes, F67084 Strasbourg, France Search for more papers by this author M.-C. Criqui Corresponding Author M.-C. Criqui Present address: Institut de Biologie Moléculaire des plantes, 12, rue du Général Zimmer, 67000 Strasbourg, France Search for more papers by this author B. Lemaitre B. Lemaitre Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du Centre National de la Recherche Scientifique, 15, rue René Descartes, F67084 Strasbourg, France Search for more papers by this author S. Uttenweiler-Joseph S. Uttenweiler-Joseph Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du Centre National de la Recherche Scientifique, 15, rue René Descartes, F67084 Strasbourg, France Search for more papers by this author L. Michaut L. Michaut Present address: Biozentrum, Department of Cell Biology, University of Basel, Klingenbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author J.-M. Reichhart J.-M. Reichhart Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du Centre National de la Recherche Scientifique, 15, rue René Descartes, F67084 Strasbourg, France Search for more papers by this author J.A. Hoffmann J.A. Hoffmann Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du Centre National de la Recherche Scientifique, 15, rue René Descartes, F67084 Strasbourg, France Search for more papers by this author D. Ferrandon Corresponding Author D. Ferrandon Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du Centre National de la Recherche Scientifique, 15, rue René Descartes, F67084 Strasbourg, France Search for more papers by this author A.C. Jung Corresponding Author A.C. Jung Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du Centre National de la Recherche Scientifique, 15, rue René Descartes, F67084 Strasbourg, France Search for more papers by this author M.-C. Criqui Corresponding Author M.-C. Criqui Present address: Institut de Biologie Moléculaire des plantes, 12, rue du Général Zimmer, 67000 Strasbourg, France Search for more papers by this author B. Lemaitre B. Lemaitre Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du Centre National de la Recherche Scientifique, 15, rue René Descartes, F67084 Strasbourg, France Search for more papers by this author S. Uttenweiler-Joseph S. Uttenweiler-Joseph Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du Centre National de la Recherche Scientifique, 15, rue René Descartes, F67084 Strasbourg, France Search for more papers by this author L. Michaut L. Michaut Present address: Biozentrum, Department of Cell Biology, University of Basel, Klingenbergstrasse 70, CH-4056 Basel, Switzerland Search for more papers by this author J.-M. Reichhart J.-M. Reichhart Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du Centre National de la Recherche Scientifique, 15, rue René Descartes, F67084 Strasbourg, France Search for more papers by this author J.A. Hoffmann J.A. Hoffmann Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du Centre National de la Recherche Scientifique, 15, rue René Descartes, F67084 Strasbourg, France Search for more papers by this author Author Information D. Ferrandon 1, A.C. Jung 1, M.-C. Criqui 2, B. Lemaitre1, S. Uttenweiler-Joseph1, L. Michaut3, J.-M. Reichhart1 and J.A. Hoffmann1 1Institut de Biologie Moléculaire et Cellulaire, UPR 9022 du Centre National de la Recherche Scientifique, 15, rue René Descartes, F67084 Strasbourg, France 2Present address: Institut de Biologie Moléculaire des plantes, 12, rue du Général Zimmer, 67000 Strasbourg, France 3Present address: Biozentrum, Department of Cell Biology, University of Basel, Klingenbergstrasse 70, CH-4056 Basel, Switzerland ‡D.Ferrandon, A.C.Jung and M.-C.Criqui contributed equally to this work The EMBO Journal (1998)17:1217-1227https://doi.org/10.1093/emboj/17.5.1217 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info A hallmark of the systemic antimicrobial response of Drosophila is the synthesis by the fat body of several antimicrobial peptides which are released into the hemolymph in response to a septic injury. One of these peptides, drosomycin, is active primarily against fungi. Using a drosomycin–green fluorescent protein (GFP) reporter gene, we now show that in addition to the fat body, a variety of epithelial tissues that are in direct contact with the external environment, including those of the respiratory, digestive and reproductive tracts, can express the antifungal peptide, suggesting a local response to infections affecting these barrier tissues. As is the case for vertebrate epithelia, insect epithelia appear to be more than passive physical barriers and are likely to constitute an active component of innate immunity. We also show that, in contrast to the systemic antifungal response, this local immune response is independent of the Toll pathway. Introduction In past years, several advances have highlighted the primordial role of innate immunity in providing a quick effector response to infections in vertebrates (Janeway, 1989; Fearon, 1997). Furthermore, natural immunity appears not only to trigger the adaptive immune response but also to direct the type of effector response in clonally selected immune cells that is appropriate to fight efficiently against infections (Medzhitov and Janeway, 1997). However, it has become clear that a first line of defense of the organism consists of the local synthesis and release of antimicrobial peptides in tissues, also called barrier epithelia, which are in direct contact with microorganisms. The role of these antimicrobial peptides is illustrated dramatically in the case of cystic fibrosis patients, where β-defensin expressed in the conducting and respiratory airway is inactivated by the high salt concentrations in the airway surface fluid due to a defect in the cystic fibrosis transmembrane conductance regulator. As a result, the airway may become colonized by microbial pathogens, and chronic inflammation ensues (Smith et al., 1996; Goldman et al., 1997). In Drosophila, a septic wound induces the rapid appearance in the hemolymph of a battery of antibacterial peptides that includes the cecropins (Kylsten et al., 1990; Tryselius et al., 1992), diptericin (Wicker et al., 1990), drosocin (Bulet et al., 1993), insect defensin (Dimarcq et al., 1994), metchnikowin (Levashina et al., 1995), attacin (Asling et al., 1995) and one major antifungal peptide, drosomycin (Fehlbaum et al., 1995). These peptides are synthesized mostly in the fat body, a functional equivalent of the liver, and secreted into the hemolymph. We will refer to this reaction as the systemic antimicrobial response. Since experimental wounds are restricted to a single point of entry and since all of the disseminated fat body is responding to the attack, it is thought that a signal is transmitted through the hemolymph from the entry site of microorganisms, where non-self recognition presumably occurs, to the fat body. It is generally appreciated today that the insect host defense presents remarkable similarities to vertebrate innate immunity, pointing to a certain degree of common ancestry (reviewed in Hoffmann et al., 1996; Hoffmann and Reichhart, 1997). In this context, we have asked whether antimicrobial peptides are also expressed in barrier epithelia in Drosophila. In this study, we address this question for the antifungal peptide drosomycin in larvae and adults of this species. To monitor the expression of drosomycin in live flies, we designed a transgenic reporter system based on the green fluorescent protein (GFP). This protein fluoresces without specific cofactors when illuminated at the right wavelengths, and can be detected in live animals provided they are reasonably transparent (Chalfie et al., 1994; Wang and Hazelrigg, 1994). Further, the fluorescence can be quantified using fluorimeters or imaging devices. Since there is no enzymatic amplification step, GFP is less sensitive as a reporter gene than the bacterial β-galactosidase gene that has been broadly used so far. However, new generations of mutant GFPs that are brighter may compensate for these shortcomings (Heim et al., 1995; Cormack et al., 1996). Indeed, we show here that S65TGFP is an adequate reporter of inducible gene expression in Drosophila. Using drosomycin–GFP reporter transgenes, we find in some non-experimentally immunized animals an expression of the drosomycin–GFP reporter gene in a variety of epithelial tissues. These observations suggest the existence of a local immune response in Drosophila in addition to the classically described systemic response induced by wounding. Thus, like vertebrate epithelia, insect epithelia may be more than passive physical barriers and are likely to constitute an active component of innate immunity. Results The drosomycin–GFP reporter gene is expressed in the fat body and hemocytes during the systemic immune response We have constructed several drosomycin–GFP reporter genes using either the wild-type or the S65T versions of GFP (Materials and methods). The RNA and protein levels of the reporter constructs induced after immunization are similar to those of the endogenous drosomycin as judged by Northern and Western blot analysis (data not shown). Furthermore, the comparison of the kinetics of protein accumulation as monitored by Western blot analysis with that of its fluorescence monitored quantitatively by fluorometry revealed that S65TGFP becomes fluorescent shortly (1–2 h) after its synthesis in the fat body of the adult (M.-C.Criqui, A.C.Jung and D.Ferrandon, unpublished data). Like the endogenous drosomycin, S65TGFP accumulates progressively during the systemic response and reaches a maximum 3 days after induction. To monitor qualitatively the expression of the drosomycin–GFP reporter gene during the systemic immune response, we looked at adult transgenic flies using a dissecting microscope equipped with an epifluorescence illumination module and the relevant set of filters. Through the cuticle, a diffuse green fluorescence was observed all over the immunized animals, whereas control animals displayed only a weak autofluorescence (Figure 1A). Upon dissection of the animal, it appeared that most of this fluorescence was due to the expression of the GFP reporter gene in the disseminated fat body of the adult (Figure 1C). No qualitative differences were noted between different lines carrying independent insertions of the transgenes. Figure 1.The drosomycin–GFP reporter gene is expressed in the fat body during the systemic response. (A) The transgenic fly shown on top has been immunized 48 h before, whereas the control fly (bottom) from the same line has not. Note the melanized scar on the thorax where the injury was performed. The fluorescence observed in the posterior part of the abdomen of the non-immunized fly corresponds to the constitutive expression of drosomycin in the sperm storage structures of the female (shown in Figure 4A). (B) The transgenic larva shown on top has been immunized for 12 h (melanized scar at the posterior end); the bottom larva is a non-immunized control. The reporter gene is expressed in the lobes of the fat body. (C) Dissected fat body lobule of an adult undergoing a systemic immune response. The expression of the reporter gene in the disseminated fat body accounts for the fluorescence observed through the cuticle of the insect (A). GFP is found in the cytoplasm and is excluded from lipidic globules. (D) Enlargement of the immunized larva shown in (B). The tubular structure that spans most of the larva is one of the two dorsal tracheal trunks. It is illuminated by the neighboring fat body tissue but is not itself fluorescent, as can be seen upon dissection. Download figure Download PowerPoint The S65TGFP reporter gene could first be detected 4–6 h after induction; the signal could usually be detected reliably in whole flies after 8 h with a dissecting scope. However, in keeping with the quantitative results described above, the fluorescence was much stronger after 24 h. In the transparent third instar larva, GFP fluorescence was detected mostly uniformly in all lobes of the fat body of immunized animals starting 4–6 h after injury, whereas usually no fluorescence could be observed in control animals (Figure 1B and D). These results agree with those obtained by in situ hybridization with a drosomycin probe (Fehlbaum et al., 1995). We also found reporter GFP expression in a subset of larval plasmatocytes and lamellocytes after a septic wound. Expression of the drosomycin–GFP reporter gene in the larval tracheal system in the absence of experimental immune challenge While looking at a large number of control non-induced larvae, we noticed that a few larvae displayed an apparently spontaneous expression of the reporter gene. The proportion of drosomycin–GFP-expressing animals was quite variable from one culture vial to the next and was probably dependent on culture conditions such as crowding, age of the culture and/or the microbial environment of the culture medium. Of these larvae, a minor percentage showed an expression in the fat body which reflected a systemic immune response due probably to natural infections. Interestingly, the reporter gene was also detected in different tissues of other larvae, especially in the tracheal epithelia. The most frequent expression pattern was observed in the anterior or posterior spiracles. This expression was often limited to one spiracle of the pair (Figure 2), suggesting a limited response to a local infection. GFP was detected in the epithelium of the tracheal trunk next to the spiracle, but not in the spiracular chamber itself. The extent of this expression was sometimes limited to a ring. In other insects, however, this spiracular expression extended to most of the tracheal dorsal trunk and transverse connectives, indicating that an aerial infection had progressed along this trachea. Rarely, some larvae expressed the reporter gene in the central but not the distal parts of the dorsal trunk. Thus, it was not always possible to correlate an expression in the tracheae with a spiracular expression. Furthermore, some insects displayed a GFP fluorescence only in the transverse connectives, lateral trunk and other smaller tracheae, but not in the dorsal trunk (Figure 2E). The expression of the reporter gene in the tracheal system was almost totally abolished in larvae reared under axenic conditions. Figure 2.Localized expression of the drosomycin–GFP reporter gene in larvae. (A) This transgenic larva was found in a culture vial and expresses the reporter gene in the right dorsal tracheal trunk in the absence of any experimentally applied immune challenge. Note that only one of the two tracheal trunks is affected. The fluorescent dot on the left corresponds to the expression of the reporter gene in the right anterior spiracle of the larva. The fluorescence is rarely found in the whole tracheal trunk and usually only spans a portion of the trunk. Since these expression patterns are not found in all larvae of a line and since all lines display expression of the reporter genes in a variable proportion of the larvae, these expressions are not the result of an enhancer-trap effect of the transposon insertions. (B) Enlargement of (A) showing that the reporter gene is expressed in the tracheal epithelium that secretes and surrounds the tracheal cuticle. The fluorescence is also found in the transverse connectives that emanate from the dorsal trunk to irrigate the tissues of the larva. (C) Anterior spiracle of a non-immunized larva observed at high magnification. The fluorescence forms a ring at the base of the spiracle. This ring, when present, sometimes extends further posteriorly along the dorsal tracheal trunk, depending on the larva. (D) Posterior spiracles of a non-immunized larva observed at high magnification. Only the distal part of the left dorsal trunk expresses the reporter gene; the right tracheal trunk is partially illuminated by the left dorsal trunk but does not itself express the drosomycin–GFP gene. A description of spiracular structures can be found in Manning and Krasnow (1993). (E) Expression of the reporter gene in the transverse connectives and the lateral branches of the tracheal system. There is no expression in the dorsal trunks through which presumably all infections should propagate. One possibility is that the cuticular sheath is thicker in the dorsal trunk and that microorganisms would break through the thinner cuticle found in the transverse connectives more easily, thus triggering a local response there. Another non-exclusive explanation we can bring forward relies on phenomena occurring during larval molts. During embryogenesis, the tracheal system originates from metameric invaginations in the epidermis (reviewed in Manning and Krasnow, 1993). These invaginations become non-functional larval spiracles that are connected to the tracheal system through the spiracular branches. During molts, the old cuticle is degraded in each metamere and is dragged out through the corresponding spiracular branches and spiracles that re-open at that time. Thus, it is possible that infections could draw profit from these events to enter the respiratory tract through the usually collapsed spiracular branch. (F and G) Expression of the reporter gene in the salivary glands of a pupa that had been dipped when at the third larval instar into a concentrated solution of Aspergillus fumigatus. This expression pattern is also observed, albeit rarely, in non-immunized larva. The salivary glands and the salivary duct can be seen through the pupa (F). All cells of the dissected salivary glands express the reporter gene at an equal level (G). Download figure Download PowerPoint To check that the GFP expression we detect in the tracheal system corresponds to the actual synthesis of the endogenous drosomycin peptide in this tissue, we dissected fluorescent and non-fluorescent parts of tracheal trunks. Strikingly, we could detect a peak corresponding to the drosomycin peptide in the fluorescent tracheae and not in the non-fluorescent ones by MALDI-TOF mass spectrometry on isolated tracheal trunks, thus demonstrating the relevance of the GFP reporter to epithelial tissue expression of drosomycin (Figure 3). Figure 3.Detection of endogenous drosomycin in fluorescent tracheae by MALDI-TOF mass spectrometry. The tracheal trunks of larvae were dissected under the fluorescence dissecting microscope. Fluorescent or non-fluorescent tracheal portions were deposited individually on different probes for mass spectrometry analysis. (A) Results obtained with a drosomycin–GFP-expressing trachea. (B) Data obtained with a non-fluorescent trachea. The peak at m/z 4910 (mass/charge) corresponds to the singly charged ions of drosomycin as controlled with recombinant drosomycin in the same conditions of analysis (Materials and methods). In some cases, the fluorescent and non-fluorescent portions of the same trachea were analyzed, and similar results obtained. Download figure Download PowerPoint Induction of the expression of the drosomycin–GFP reporter gene following an exposure to microbial agents To show that the localized expression patterns of the drosomycin–GFP reporter gene in the respiratory system are indeed a local response to a microbial infection, we dipped early third instar larvae in concentrated solutions of various bacteria or fungal spores for 30 min. The larvae were then allowed to recover from the treatment for 5–7 days at 18°C in a normal fly vial. In a typical experiment, 80% of the larvae that had been treated with the Gram-negative bacteria Erwinia carotovora carotovora developed a strong expression of the reporter gene throughout the tracheal system. In contrast, only 10% of the control animals that had been dipped in water displayed an expression of the transgene that was limited to the spiracles. In a few experiments, the proportion of control animals reacting to water treatment was somewhat higher than 10%, yet transgene expression was limited to the spiracles and did not propagate to the whole tracheal system. These experiments show that drosomycin can be synthesized in the tracheal system in response to a microbial infection. Other expression patterns of the drosomycin–GFP reporter gene in larvae and adults in the absence of experimental immune challenge A few larvae and pre-pupae were found occasionally to express the reporter gene in all cells of the salivary glands (Figure 2F and G). In contrast, all transgenic adults carrying the drosomycin–GFP reporter displayed some fluorescence in the terminal coiled regions of the salivary glands (Figure 4E). The intensity of fluorescence was variable from one fly to the next. We may assume that this expression is weakly constitutive and can be induced strongly. Often, two paired fluorescent patches were seen inside the proboscis; they most likely correspond to expression of the drosomycin–GFP reporter gene in the two small labellar glands whose secretory ducts open at the beginning of the alimentary canal (Figure 4C). Very few individuals displayed expression of the reporter gene in the pseudotracheae of the proboscis (Figure 4D). The salivary and labellar gland expression was sometimes observed already in late pupal stages. Figure 4.Localized expression of the drosomycin–GFP reporter gene in adults. (A) Constitutive expression of the reporter gene in the seminal receptacle (arrow) and spermathecae (arrowheads) of an immunized adult. The fluorescence in the sperm storage structures is much brighter than that of the surrounding fat body and is probably due to the continuous accumulation of the reporter protein in these structures. The second spermatheca is not in the focal plane of the picture. (B) Expression of the reporter gene in the male reproductive tract. In this non-immunized male, the fluorescence is found in the anterior ejaculatory duct (arrow), the ejaculatory bulb (arrowhead) and the posterior ejaculatory duct (not shown). The extent of the expression in the ejaculatory bulb is variable and, when present, may be limited to the posterior part of the ejaculatory duct. The yellow fluorescence observed in the tip of the Malpighi tubules marked by an * is due to the autofluorescence of these structures. (C) Expression of the drosomycin–GFP reporter in the labellar glands. The fluorescence is found inside the labellum in paired structures. This expression pattern is frequent. (D) High magnification of the bottom of a labellum. Drosomycin–GFP expression is found in the pseudotracheae that conduct liquid food to the tip of the labrum. (E) Strong expression of the reporter gene in the adult salivary gland. The fluorescence is limited mostly to the terminal coiled portion of the salivary gland. (F) Expression of the drosomycin–GFP in epidermal cells of the maxillary palp. The expressing cells might be sensilla basiconica, i.e. chemoreceptors for taste and smell. In other adults, the fluorescence in the maxillary palp appears more diffuse and may be due to expression in the maxillary palp trachea. (G) Expression of the reporter gene in the abdominal tracheal system observed through the cuticle. The fluorescence starts at the abdominal spiracle and extends to the tracheal system. (H) Expression of the reporter gene in abdominal sets of cells. Two groups of three cells express drosomycin–GFP at the posterior region of each tergite. Other groups of non-identified cells on the head and ventral abdomen are not shown (see text). Download figure Download PowerPoint The above results raise the possibility that drosomycin is secreted into the alimentary canal by two sets of secretory organs, the salivary and labellar glands. Some adults exhibited other expression patterns in the head. For instance, fluorescence was detected fairly often in the maxillary palps. In some cases, it was clear that expression was taking place in the epithelial cells (Figure 4F), whereas in others, the fluorescence appeared more diffuse and could possibly be due to drosomycin–GFP reporter expression in the small maxillary palp trachea. Similar observations were made in the antennal segments. As in larvae, expression of the drosomycin–GFP reporter gene was sometimes detected in the adult respiratory system, usually of older insects (Figure 4G). Fluorescence was observed in the spiracles, abdominal tracheae, leg tracheae, the head air sacs and tracheae, and probably in the thoracic air sacs. However, the expression in most cases did not extend to the whole respiratory system, but only portions thereof, suggesting a local immune response in the trachea following an aerial infection. Occasionally, drosomycin–GFP could be detected in the ejaculatory duct, and more rarely in the ejaculatory bulb of some males (Figure 4B). Depending on the fly, the expression in the ejaculatory duct extended more or less anteriorly. These observations suggest that infections can propagate along the genital ducts and induce a local synthesis of drosomycin to fight off this infection. In a few adults, several rows of three cells beneath the dorsal abdominal cuticula were found to express the drosomycin–GFP reporter gene (Figure 4H). This expression pattern also included two thin continuous rows of epidermal cells that partially surround the dorsal part of the eyes, as well as two paired groups of abdominal cells that lie on the ventral side at the frontier with the thorax. To our knowledge, these groups of cells have not been identified so far. All the expression patterns recapitulated in Table I were never induced by wounding experiments that trigger the systemic response. Table 1. Local expression of the drosomycin–GFP reporter transgene in the absence of experimentally applied immune challenge Tissue Inducible expressiona Constitutive expressionb Larval respiratory system Tracheal epithelium ** Spiracles *** Larval digestive system Salivary glands * Adult respiratory system Tracheal epithelium ** Spiracles ** Adult digestive system Salivary glands *** # Labellar glands ** Pseudotrachea * Crop * Rectal ampulla * Adult cuticle Maxillary palps ** Antenna * Rows of three abdominal cellsc * Reproductive system ♀ spermathecae and seminal receptacle ? ### ♂ ejaculatory duct * Systemic expression Fat body * a The frequency of each expression is scored semi-quantitatively from to . However, these should be considered only as indications, since the actual frequencies vary considerably and are dependent on the vial culture conditions, rendering a statistical analysis meaningless. b The strength of the constitutive expression is indicated: # weak expression; ### strong expression, the intensity of which is stronger than that of the systemic expression in the fat body. c These non-identified group of cells are shown in Figure 4H and described in the Results. The drosomycin–GFP transgene reveals a constitutive expression of drosomycin in the female sperm storage organs All adult females carrying the drosomycin–GFP reporter gene in the different transgenic lines displayed a bright source of fluorescence that could be seen through the cuticle of the posterior part of the abdomen. Upon dissection, the fluorescence was found in the female sperm storage structures: the two spermathecae and the seminal receptacle (Figure 4A). In keeping with this expression, endogenous drosomycin RNA was also detected by in situ hybridization in these organs (L.Michaut and R.Lanot, unpublished observations). This signal is not dependent on copulation since it was also found in virgin females. Further, it was present in females grown under axenic conditions. We conclude that this expression is constitutive. Genetic analysis: a pathway other than Toll? The systemic antifungal response in the adult has been shown to be under the control of the spätzle/Toll/tube/pelle/cactus pathway (Lemaitre et al., 1996). The inducible expression of drosomycin is abolished in spätzle (spz), recessive lack-of-function Toll (Tl), tube (tub) and pelle (pll) mutants. Drosomycin is constitutively expressed in cactus (cact) and Tl dominant gain-of-function mutants (Lemaitre et al., 1996). Most of this gene cassette, which also establishes the primary dorso-ventral pattern (reviewed in Morisato and Anderson, 1995), has been conserved during evolution and appears to control the activation of the NF-κB transcription factor in the vertebrate inflammatory responses (Ingham, 1994; Kopp and Ghosh, 1995; Verma et al., 1995; Baeuerle and Baltimore, 1996). When this pathway is altered, flies succumb to a fungal, but not to a bacterial infection (Lemaitre et al., 1996). We therefore investigated whether the local expression of drosomycin that is detected in several tissues of ectodermal origin is also controlled by this pathway.