spätzle

DEVELOPMENTAL BIOLOGY

Embryonic

Unprocessed Spätzle protein is present in perivitelline fluid prior to fertilization. The 23 kd proteolyzed form of Spätzle is first detected one hour following egg laying, at the time when Toll is first active in promoting Dorsal protein nuclear localization (Morisato, 1994).

Dorsal-ventral polarity of the Drosophila embryo is established by a nuclear gradient of Dorsal protein, generated by successive gurken-Egfr and spätzle-Toll signaling. Overexpression of extracellular Spätzle dramatically reshapes the Dorsal gradient: the normal single peak is broadened and then refined to two distinct peaks of nuclear Dorsal, to produce two ventral furrows. This partial axis duplication, which mimics the ventralized phenotype caused by reduced gurken-Egfr signaling, arises from events in the perivitelline fluid of the embryo and occurs at the level of Spätzle processing or Toll activation. The production of two Dorsal peaks is addressed by a model that invokes the action of a diffusible inhibitor, which is proposed to normally regulate the slope of the Dorsal gradient (Morisato, 2001).

The Dorsal gradient in the wild-type embryo possesses a characteristic shape. The domain of peak nuclear Dorsal in the embryo can vary over a wide range, depending on the level of Spätzle production, but never exceeds the limits presaged by the expression of pipe in the ovary. In contrast, the slope of the Dorsal gradient, as measured by the extent of sog expression, is relatively constant under these conditions (Morisato, 2001).

The shape of the Dorsal gradient is dramatically changed in embryos laid by females carrying mutations in the gurken-Egfr signaling pathway. Not only do these embryos expand Twist expression, as a consequence of a reduction in the dorsalizing signal that establishes egg chamber asymmetry, but they exhibit two distinct peaks within the Twist domain that give rise to two ventral furrows. In the experiments described here, this partial axis duplication is not evident during oogenesis, because pipe RNA was found to be expressed in a single broad domain in follicle cells. The production of two Dorsal peaks could be mimicked by injecting high levels of spz RNA into the pre-cellular embryo cytoplasm, suggesting that pattern refinement occurs during embryogenesis. It is suggested that while the size of the ventral domain is expanded in grk and Egfr ovarian egg chambers, the partial axis duplication observed in mutant embryos is caused by reactions occurring later in the embryo (Morisato, 2001).

It may have been easier to imagine how the selection of one or two gradient peaks would involve signaling within the follicular epithelium, because spatial information could then be stably maintained and transmitted by cells. The elaboration of the two dorsal appendages in the Drosophila eggshell results from a series of such intercellular signaling events. Activation of Egfr by Gurken stimulates transcriptional induction of Argos, a secreted Egfr inhibitor, which then downregulates Egfr activity in the initial central domain, leaving two lateral domains of signaling (Morisato, 2001).

In fact, the findings described in this paper argue that events involving the diffusion of an extracellular morphogen not only regulate the gradient slope, but perhaps unexpectedly, determine the position and number of maxima within the axis in response to the broad cues generated during oogenesis. Reaction-diffusion models have been applied to analyze the respective contributions of the gurken-Egfr and spätzle-Toll pathways in generating embryonic pattern. The current studies provide experimental support for this theoretical work, and present opportunities for understanding the underlying mechanisms (Morisato, 2001).

Formation and maintenance of the Dorsal gradient appear dynamic. The shape of the Dorsal gradient in the wild-type embryo does not change markedly after nuclear translocation is first detected. In embryos laid by grk females or embryos expressing high levels of Spätzle, however, the shape of the Dorsal gradient is subtly modified. In particular, the minimum lying between the two Dorsal peaks becomes deeper in older embryos. This observation suggests that signaling takes place over a period of time, and explains how an initial asymmetry, in the form of the broad stripe of pipe, might be gradually refined into a gradient of positional information (Morisato, 2001).

Easter cleavage generates N-terminal and C-terminal processed Spätzle forms. Although C-terminal processed Spätzle has been shown to be directly required for activating Toll, no activity has been ascribed to N-terminal Spätzle. Could N-terminal Spätzle act as an inhibitor of signaling? In the embryo, alternative splicing of the spz gene results in the production of a number of proteins that diverge in the N terminus, while sharing a common C terminus containing the cystine knot motif. When embryonic extracts are probed with anti-N-Spätzle antibodies, several proteins corresponding to the N-terminal products of the Spätzle processing reaction are identified. When embryonic extracts are probed with anti-C-Spätzle antibodies, a single processed form is observed. This C-terminal processed Spätzle is present at much higher levels in embryos lacking Toll. The lack of an effect on the level of N-terminal Spätzle by Toll suggests that the products of the cleavage reaction are not physically associated and undergo different fates. Consistent with this interpretation, when N-terminal Spätzle is affinity purified from embryonic extracts, cleaved C-terminal Spätzle is not co-purified. These observations raise the possibility that the N- and C-terminal forms have different functions in signaling (Morisato, 2001).

Evidence is presented for the following model, which accounts for many of the observations described above. The initial shape of the gradient (at t₀) is established by the proteolytic activation of Spätzle in a relatively broad domain, reflecting the ventral region of the egg chamber that expresses pipe RNA. It is proposed that the Spätzle processing reaction generates an inhibitor that negatively regulates the production of the ventral signal, possibly at the level of Easter protease activity or the interaction between processed Spätzle and Toll. Whereas processed C-terminal Spätzle is believed to bind to Toll quickly and show limited movement after cleavage, it is postulated that the hypothetical inhibitor undergoes broader diffusion. In the wild-type embryo, inhibitor action is responsible for establishing the region of high nuclear Dorsal, corresponding to the Twist domain, to be narrower than the ventral region of the egg chamber expressing pipe RNA. The final shape of the Dorsal gradient (at t₁) is generated over time by the opposing effects of processed Spätzle and the inhibitor (Morisato, 2001).

In embryos produced by grk females, it is inferred that Spätzle processing is occurring at wild-type levels, but the reaction is distributed over a broader domain. The ventral region becomes sufficiently expanded such that the difference between the diffusion rates of processed Spätzle and the inhibitor can reshape the ventral domain itself. In particular, rapid diffusion of the inhibitor results in a lower concentration at each border, compared with the center of the domain. This change in the ratio of processed Spätzle to inhibitor eventually produces a peak at each border of the expanded domain. By this reasoning, an expanded ventral domain never generates more than two peaks because there are never more than two borders (Morisato, 2001).

Embryos synthesizing high levels of precursor Spätzle increase the amount of processed Spätzle, thereby expanding the domain of high nuclear Dorsal. In contrast to embryos produced by grk females, where a wild-type level of processed Spätzle is distributed over a broader area, an increased level of processed Spätzle appears to generate a broader domain in these injected embryos. Pattern refinement is observed only at the highest levels of Spätzle production, perhaps because only in this situation can the minimum domain size be created (Morisato, 2001).

The complexity of the patterning process is underscored by the observation that partial axis duplication can be induced by both an increase and decrease in spz dosage, depending on the extent of pipe expression dictated by gurken-Egfr signaling. A deeper understanding of this dynamic behavior will probably require the application of mathematical approaches (Morisato, 2001).

In order to explain the production of two Dorsal peaks, the inhibitor must be generated in a spatially asymmetric manner. In the model outlined here, inhibitor production has been linked to the proteolytic processing of Spätzle to satisfy this condition. The results described above raise the possibility that N-terminal processed Spätzle is acting as an inhibitor to shape the Dorsal gradient, although the model does not exclude action of other negative regulators. For example, pattern refinement may involve maternal Dpp signaling, acting parallel or downstream of Toll, which has been shown to reduce the magnitude of Dorsal nuclear translocation. At the mechanistic level, the N-Spätzle inhibitor could be recruiting molecule X, found in a stable complex with Easter and suggested to be a serpin, or it could be acting on one of the proteases that act upstream of Easter. Alternatively, the inhibitor could be negatively regulating the binding of processed Spätzle to Toll (Morisato, 2001).

The strongest genetic support for diffusion playing a role in the formation of the Dorsal gradient comes from mosaic analysis. Ventral pipe- clones not only result in the absence of Twist expression, but also produce a corresponding loss of sog expression in lateral regions of the embryo. These results rule out the presence of a pre-existing gradient in the follicular epithelium. The requirement for a ventral source of the Dorsal gradient suggests that the slope is generated by the diffusion of a component in the embryonic signaling pathway, although a sequential induction mechanism within the follicular epithelium is not formally excluded (Morisato, 2001).

An output dependent on the ratio of the respective diffusion rates of activator and inhibitor, rather than diffusion of the activator alone, may allow the embryo to generate a more stable gradient shape in response to the broad spatial signals defined during oogenesis. Moreover, such a mechanism may help the embryo cope with changes in perivitelline fluid viscosity, caused by fluctuations in temperature and humidity after egg deposition, that would otherwise result in developmental defects. Coupling diffusion of an activator and inhibitor may represent a general strategy for regulating extracellular signaling in other patterning reactions (Morisato, 2001).

Retromer promotes immune quiescence by suppressing Spatzle-Toll pathway in Drosophila

The Toll and Toll-like receptor signaling pathways are evolutionarily conserved pathways that regulate innate immunity in insects and mammals. While efforts have been made to clarify the signal transduction events that occur during infection, much less is known about the components that maintain immune quiescence. This study shows that retromer, an intracellular protein complex known for regulating vesicle trafficking, functions in modulating the Toll pathway in Drosophila melanogaster. In mutant animals lacking retromer function, the Toll pathway but not JAK-STAT or IMD pathway is activated, triggering both cellular and humoral responses. Genetic epistasis and clonal analysis suggest that retromer regulates a component that acts upstream of Toll. The data further show that in the mutant the Toll ligand Spatzle has a processing pattern similar to that of after infection. Together, the results suggest a novel function of retromer in regulating Toll pathway and innate immunity at a step that modulates ligand processing or activity (Zhou, 2014).

Based on previous knowledge that retromer regulates trafficking of transmembrane proteins, one can envisage that retromer normally may regulate the Toll pathway in one of the four following ways: (1) in Toll-responsive cells to transport the Toll-Spz complex for destruction; (2) in Spz secreting cells to suppress the release of active Spz; (3) in certain cells to assist the clearance of active Spz in the hemolymph; or (4) in certain cells to repress Spz through an indirect effect of other yet to be indentified components. In the Vps35 mutant clonal cells in fat bodies no increased Dorsal nuclear localization was observed, indicating that retromer is not simply regulating the Toll pathway cell-autonomously. Epistasis analysis suggests that retromer acts between Toll and SPE. Even though Spz is the only known component in between, there can be many other proteins that regulate the processing, maturation, trafficking or degradation of Spz in normal flies in order to restrict the activity of the Toll signaling pathway prior to infections. The full mechanism of Spz maturation is not yet unveiled and the retromer function in this process requires further investigation. Although the possibility cannot be excluded that retromer has an indirect effect on Spz, a function of retromer in anti-release and/or clearance of active Spz is favored. Retromer has been shown to target transmembrane proteins. It will be intriguing to identify the transmembrane target of retromer in the context of regulating Spz and explore the mechanism of how this transmembrane target suppresses the release and/or assists the clearance of active Spz. Equally important is to examine whether modulating retromer-dependent Spz maturation is part of the activation mechanism of the Toll pathway during infections (Zhou, 2014).

This study has identified a role of retromer in negatively regulating the Toll pathway to maintain immune quiescence. In the absence of infection, the loss of retromer activity alone is capable of activating the Toll pathway and launching both the cellular and humoral immune responses. Furthermore, genetic epistasis and mosaic analysis suggest that retromer acts upstream of Toll and downstream of Spatzle-Processing Enzyme (SPE), and a retromer function was uncovered in restricting the processing/maturation of Spz. In summary, retromer plays a critical role in suppressing the auto-activation of the innate immune system through Spz in the Toll pathway in Drosophila (Zhou, 2014).

An ancient defense system eliminates unfit cells from developing tissues during cell competition

Developing tissues that contain mutant or compromised cells present risks to animal health. Accordingly, the appearance of a population of suboptimal cells in a tissue elicits cellular interactions that prevent their contribution to the adult. This study reports that this quality control process, cell competition, uses specific components of the evolutionarily ancient and conserved innate immune system to eliminate Drosophila cells perceived as unfit. Toll-related receptors (TRRs) and the cytokine Spatzle (Spz) lead to NFκB-dependent apoptosis. Null mutations in Toll-3, Toll-8, or Toll-9 suppress elimination of loser cells, increasing loser clone size and cell number per clone, but do not alter control clones. Diverse 'loser' cells require different TRRs and NFκB factors and activate distinct pro-death genes, implying that the particular response is stipulated by the competitive context. These findings demonstrate a functional repurposing of components of TRRs and NFkappaB signaling modules in the surveillance of cell fitness during development (Meyer, 2014).

Altogether, these results demonstrate that the conceptual resemblance between cell competition and innate immunity is matched with genetic and mechanistic similarities. Thus, cells within developing tissues that are recognized as mutant or compromised are competitively eliminated via a TRR- and NFκB-dependent signaling mechanism. Although similar core signaling components are activated in both processes, cell competition culminates in local expression of proapoptotic genes rather than systemic induction of antimicrobial genes. Because cell competition is initiated by the emergence of cells of different fitness than their neighbors in a tissue, it is surmised that the initiating signal is common to many competitive contexts. The genetic data leads to a proposal of a model for how this signal is detected and transduced. The results point to a role for Spz in signal detection, as it is a secreted protein that is required for the killing activity of competitive conditioned medium (cCM), is a known ligand for the Toll receptor, and is produced by several tissues in the larva. Thus, it is speculated that Spz functions as a ligand for one or more TRR in cell competition. Because Spz must be activated through a series of proteolytic steps, the relevant proteases may respond directly to the initiating signal in cell competition. It is proposed that the genetic identity or context of the competing populations influences activation of different TRR signaling modules and that the precise configuration of TRRs on loser cells dictates which of the three Drosophila NFκB proteins is activated. How signaling to the NFκBs is restricted to the loser cells is not known, but higher expression of Toll-2, Toll-8, and Toll-9 in loser cells could bias signal transduction. PGRP-LC, a receptor known to bind only bacterial products, also plays a role in Myc-induced competition. As commensal gut microflora is known to influence larval growth, this raises the possibility that it also contributes to the competitive phenotype (Meyer, 2014).

Throughout evolution, signaling modules have adapted to fulfill different functions even within the same species. This study has provided evidence for adaptation of TRR-NFκB signaling modules in an organismal surveillance system that measures internal tissue fitness rather than external stimuli. It is noteworthy that the killing of WT cells by supercompetitor cells is a potentially pathological form of cell competition that could propel expansion of premalignant tumor cells. If so, activated TRR-NFκB signaling modules in nonimmune tissues could be diagnostic markers, and their competitive functions could serve as therapeutic targets for cancer prevention (Meyer, 2014).

Effects of Mutation or Deletion

A dominant Toll product promotes ventral development even in the absence of spz; therefore spz acts upstream of Toll. spz dominant females produce ventralized embryos, and spz/Toll minus heterozygote females are strongly dorsalized. This means that spz is responsible for the ventralized phenotype (Moresato, 1994).

The Toll signaling pathway functions in several Drosophila processes, including dorsal-ventral pattern formation and the immune response. This pathway is required in the epidermis for proper muscle development. Because Toll mutations affect the development of all 30 muscle fibers in each hemisegment, and not just the several that express Toll, or those closest to the CNS, it semed likely that the epidermal expression is most relevant to muscle development. In the epidermis, Toll expression is highest in the epidermal muscle attachment (EMA) cells, aligned along the segment border; these cells are known to play an important role in muscle patterning. The zygotic Toll protein is necessary for normal muscle development; in the absence of zygotic Toll, close to 50% of hemisegments have muscle patterning defects consisting of missing, duplicated and misinserted muscle fibers (Halfon, 1998).

The requirements for easter, spatzle, tube, and pelle, all of which function in the Toll-mediated dorsal-ventral patterning pathway have now been analyzed. spatzle, tube, and pelle, but not easter, are necessary for muscle development. Mutations in these genes give a phenotype identical to that seen in Toll mutants, suggesting that elements of the same pathway used for Toll signaling in dorsal-ventral development are used during muscle development. By expressing the Toll cDNA under the control of distinct Toll enhancer elements in Toll mutant flies, the spatial requirements for Toll expression were examined during muscle development. Expression of Toll in a subset of epidermal cells that includes the epidermal muscle attachment cells, but not Toll expression in the musculature, is necessary for proper muscle development. A 6.5-kb enhancer element drives expression solely in mesodermally derived tissues. A 1.4-kb enhancer drives expression in the epidermis, CNS midline, gut, salivary glands, Malpighian tubules, pharynx and esophagus, but not in mesodermal tissues. These two enhancers were used to drive expression of Toll in transgenic flies. The 1.4-kb enhancer express Toll in the epidermis (in a narrow strip of cells that includes the EMA cells) as well as in a cluster of cells in the lateral, mid-bodywall region of each segment. This lateral region contains the cells where the lateral transverse muscle fibers have their insertions. Flies with 1.4-kb enhancer driven Toll expression show complete rescue of the muscle error phenotype. These results suggest that signals received by the epidermis early during muscle development are an important part of the muscle patterning process (Halfon, 1998).

Although loss of single minded, a regulator of the Toll pathway in the central midline, causes positioning and insertion errors in a group of the most ventral muscles, these defects are qualitatively different from those observed in Toll mutants. The errors due to sim mutation are thus not likely due to loss of Toll expression; Toll expression in the midline appears to be uninvolved in muscle patterning. It is known that signaling from the muscle fibers induces the expression of beta1-tubulin in the EMA cells and regulates the maintenance of expression of other attachment site-specific genes such as delilah, groovin, and stripe. The nature of the Toll muscle phenotype (most of which consists of duplicated and deleted muscle fibers) suggests that Toll may be acting early in the development process, during the time of founder specification or early muscle fiber growth. The remaining errors (those in muscle insertion) may be either early or late in origin: they may be secondary to mis-specification of muscle identity (early), or alternatively, might indicate a further requirement for Toll during the insertion process (late) (Halfon, 1998).

The receptor Toll, intracellular components of the dorsoventral signaling pathway, Tube, Pelle, and Cactus, and the extracellular Toll ligand, Spätzle, but not the NF-kappaB related transcription factor Dorsal, control expression of the antifungal polypeptide gene Drosomycin in adults. Mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. In contrast, drosomycin gene induction is not affected in mutants deficient in gastrulation defective, snake and easter, all upstream of spätze in the dorsoventral pathway. The involvement of Cactus in the drosomycin induction pathway is unexpected, since, in contrast with cat, pll, tub, and Tl, the spz mutant shows no striking zygotic phenotype. The partner of Cact in the drosomycin induction pathway has not yet been identified, but it is probably a member of the Rel family, possibly Dif (Lemaitre, 1996).

There are a number of different controls on the expression of the antifungal polypeptide gene drosomycin in adults: the receptor Toll, intracellular components of the dorsoventral signaling pathway (Tube, Pelle, and Cactus), and the extracellular Toll ligand, Spätzle, but not the NF-kappaB related transcription factor Dorsal. Mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. In Tl-deficient adults, the cecropin A and, to a lesser extent, attacin, drosomycin and defensin genes are only minimally inducible, in contrast with the diptericin and drosocin genes, which remain fully inducible in this context. The drosomycin gene induction is not affected in mutants deficient in gastrulation defective, snake and easter, all upstream of spätzle in the dorsoventral pathway. The involvement of Spätzle in the drosomycin induction pathway is unexpected, since, in contrast with cat, pll, tub, and Tl, the spz mutant shows no striking zygotic phenotype. The partner of Cact in the drosomycin induction pathway has not yet been identified, but it is probably a member of the Rel family, possibly Dorsal-related immunity factor (Lemaitre, 1996).

Microarray studies have shown recently that microbial infection leads to extensive changes in the Drosophila gene expression program. However, little is known about the control of most of the fly immune-responsive genes, except for the antimicrobial peptide (AMP)-encoding genes, which are regulated by the Toll and Imd pathways. Oligonucleotide microarrays have been used to monitor the effect of mutations affecting the Toll and Imd pathways on the expression program induced by septic injury in Drosophila adults. Toll and Imd cascades were found to control the majority of the genes regulated by microbial infection in addition to AMP genes and are involved in nearly all known Drosophila innate immune reactions. However, some genes controlled by septic injury were identified that are not affected in double mutant flies where both Toll and Imd pathways are defective, suggesting that other unidentified signaling cascades are activated by infection. Interestingly, it was observed that some Drosophila immune-responsive genes are located in gene clusters, which often are transcriptionally co-regulated (De Gregorio, 2002).

To identify the target genes of the Toll and Imd pathways in response to microbial infection, the gene expression programs induced by septic injury have been compared in wild-type and mutant adult male flies using oligonucleotide microarrays. In parallel, the survival rate and the expression level of various AMP genes have been monitored after infection by various microorganisms. For the Toll pathway, a strong homozygous viable allele of spz (rm7) was selected. The spz, Tl and pll mutations, alone or in combination with rel, have similar effects on both the survival rate and pattern of AMP gene expression after microbial infection. These findings suggest that the effects of spz mutation on the transcription program induced by infection reflect the role of the entire Toll pathway in the immune response. For the Imd pathway, a null viable allele of relish (E20) was selected. Similarly to the Toll pathway, previous comparative studies did not reveal any striking difference between mutations in relish and null mutations in the genes encoding the other members of the Imd pathway such as kenny, ird5 and dredd, with the sole exception of mutations in dTAK1, which have a slightly weaker phenotype. Again, these data suggest that the effects of rel mutation on the immune response reflect the role of the whole Imd pathway. However, other pathways, including Toll, cannot be excluded from having a minor role in Relish activation (De Gregorio, 2002).

The septic injury experiments were performed using a mixture of Gram-positive and Gram-negative bacteria. This type of infection activates a wide immune response and allows the simultaneous analysis of several categories of immune-responsive genes. However, it has been shown that Toll and Imd pathways are activated selectively by different classes of microorganisms; thus, the use of a bacterial mixture might increase the redundancy of the two pathways in the control of common target genes (De Gregorio, 2002).

The microarray analysis demonstrates that the functions of Toll and Imd pathways in Drosophila immunity can be extended beyond the regulation of AMP genes. The majority of the Drosophila immune-regulated genes (DIRGs) are affected by the mutations in the Toll or Imd pathways. Many of these genes are unknown (see www.fruitfly.org/expression/immunity/ for a complete list); others can be assigned to several immune functions. The susceptibility of the Imd and Toll pathway mutants to different types of microbial infection suggested a dual aspect to the control of the antifungal response by the Toll pathway: a major role for the Toll pathway for the response to Gram-positive bacteria with a minor contribution of Imd, and a predominant role of Imd with a minor contribution of Toll to the resistance against Gram-negative bacteria. In agreement, microarray analysis shows that the Toll pathway controls most of the late genes induced by fungal infection and cooperates with the Imd pathway for the control of genes implicated in several immune reactions such as coagulation, AMP production, opsonization, iron sequestration and wound healing. Interestingly, defensin, which encodes the most effective antimicrobial peptide directed against Gram-positive bacteria, is co-regulated by both the Imd and Toll pathways. The hierarchical cluster analysis of the expression profiles combining the effect of the mutations after septic injury with the response to fungal infection provides a wealth of information that may help to elucidate the function of some of the uncharacterized DIRGs. Until now, the increased susceptibility to infection of Imd- or Toll-deficient flies has been attributed to the lack of expression of AMP genes, and it has been shown recently that the constitutive expression of single AMP genes in imd;spz double mutant flies can increase the survival rate of some types of bacterial infection. The finding that the Toll and Imd pathways are the major regulators of the Drosophila immune response now suggests that other immune defence mechanisms might contribute to the increased susceptibility to infection displayed by mutant flies (De Gregorio, 2002).

The interactions between the Toll and Imd pathways are more complex than merely regulating the same target genes. In agreement with Northern blot analysis, it has been shown that the transcriptional control of relish in response to infection receives a modest input from the Toll pathway, revealing an additional level of interaction between the two cascades. The activation of Toll may increase the level of Relish to allow a more efficient response to bacterial infection. This finding is in agreement with previous observations showing that in mutants where the Toll pathway is constitutively active (Tl^10b), all the antibacterial peptides genes, including diptericin, are induced with more rapid kinetics than in wild-type flies. Furthermore, the higher susceptibility to E.coli infection of the rel,spz double mutant compared with the rel single mutants flies indicates that Toll also has a direct, Relish-independent effect on the resistance to infection by Gram-negative bacteria. Northern blot analysis shows that relish induction in response to infection is significantly reduced in dTAK1 and dredd mutants, indicating that the Imd pathway undergoes autoregulation. Interestingly, the Imd pathway can influence the Toll pathway through the control of PGRP-SA, which encodes a recognition protein essential for the activation of the Toll pathway by Gram-positive bacteria. Again, it is interesting to notice that this interaction between the Toll and Imd pathways correlates with the contribution of both pathways to fight infection with Gram-positive bacteria. Interestingly, all the genes encoding components of the Toll pathway required for both antibacterial and antifungal responses (necrotic, spaetzle, Toll, pelle, cactus and Dif) are not controlled by the Imd pathway and are subjected to autoregulation (De Gregorio, 2002).

The Rel/NF-kappaB proteins Dif, Dorsal and Relish, which are the transactivators induced by the Toll and Imd pathways, bind to the kappaB sites present in the promoters of target genes, such as AMP genes, regulating their expression. Therefore, the analysis of the promoters of the DIRGs controlled by Toll or Imd pathways could help to identify all the direct NF-kappaB targets during infection. However, some of the effects of mutations affecting the Toll or Imd pathways that were monitored by microarray analysis might be mediated by the regulation of other transcription factors or signaling cascades. It has been shown recently in larvae that the Tep1 gene is regulated by the JAK-STAT pathway and can be activated by the Toll pathway, suggesting that Toll can control, at least partially, the JAK-STAT cascade. Two genes encoding components of the JNK pathway (puc and d-Jun) are partially regulated by Toll and Imd in response to septic injury (De Gregorio, 2002).

The presence of DIRGs independent of or only partially dependent on both the Imd and Toll pathways suggests the presence of other signaling cascades activated after septic injury. Potential candidates are MAPK and JAK-STAT pathways. Beside their developmental functions, the MAPK pathways have been implicated in wound healing (JNK) and the stress response (MEKK). The JAK-STAT pathway controls the Drosophila complement-like gene TepI. The stimuli that trigger these cascades are not known and it is not clear if these cascades are activated by exogenous or host factors. Interestingly, in vertebrates, the JAK-STAT pathway is activated by cytokines during the immune response. The microarray analysis of mutants in these pathways might help to reveal their exact contribution to the Drosophila immune response. The observation that Toll and Imd pathways control most of the DIRGs raises the question of whether these two pathways are the sole signaling cascades directly activated by microbial elictors, while the other signaling pathways are triggered by other stimuli associated with infection such as wound, stress, cytokine-like factors and Toll and Imd activities (De Gregorio, 2002).

In vertebrates, many genes involved in the immune response are grouped in large chromosomal complexes. The recent completion of the Drosophila genome did not reveal any striking chromosomal organization beside clustering of genes belonging to the same family, probably reflecting recent duplication events. In this study, it was observed that some of the genes responding to microbial infection are located in the same cytological region or are associated in transcriptionally co-regulated genomic clusters. Interestingly, microarray analysis of circadian gene expression in Drosophila has led to the identification of similar clusters of genes. Other microarray analyses might reveal the importance of the genome organization in the definition of adequate transcription programs in response to a variety of stimuli (De Gregorio, 2002).

Depletion of Serpin-27A (Spn27A) from the hemolymph following immune challenge is best explained by assuming that it binds to a cognate protease and that the resulting complex is either removed from circulation or degraded. Two intracellular signaling pathways control the expression of challenge-induced genes in the fat body, the predominant immuno-responsive tissue of Drosophila. DNA microarray data indicate that the infection-induced expression of several putative prophenoloxidase-activating enzyme (PPAE) genes in Drosophila is under the control of the Toll pathway. Moreover, Spn27A transcription is also regulated in an immune-dependent manner. It was of interest to examine whether the melanization observed in the Spn27A^ex32 mutants could be correlated to the well-defined Toll-mediated host response in Drosophila. In Dif or spaetzle (spz) mutants, in which the Toll pathway is blocked, the serpin is not removed from the circulating hemolymph. Interestingly, the serpin is removed in kenny (key) mutants that block the Imd pathway, which is not involved in the control of PPAE gene expression (De Gregorio, 2002a). A testable prediction that can be derived from these results is that in a Toll pathway mutant background PO enzymatic activity following bacterial challenge should be at very low levels. It as indeed observed that Dif- and spz-infected flies have a PO activity comparable to the basal level of non-challenged wild-type flies in contrast to key-infected flies, which show normal PO levels. The data furthermore imply that the protease (or the factor that triggers its activation), which removes Spn27A, may not be present as a zymogen activated by infection, but may need de novo protein synthesis dependent on Toll signal transduction. This was confirmed by infecting wild-type flies with bacteria in the presence of an inhibitor of translation (cycloheximide). Protein synthesis following bacterial infection was examined by mass spectrometry; none of the induced antimicrobial peptides were synthesized in the experimental conditions indicating that protein synthesis was efficiently blocked. Importantly, it was noted that in these flies during the same infection procedures the serpin was not removed from circulation. Taken together, these results on the requirement of Toll signaling pathway and protein synthesis for serpin removal show that the protease(s) removing Spn27A from circulation is synthesized de novo in response to Toll signaling. Alternatively, a component that activates it is dependent on Toll signaling-driven de novo protein synthesis (Ligoxygakis, 2002a).

Drosophila Sex-peptide stimulates female innate immune system after mating via the Toll and Imd pathways

Insect immune defense is mainly based on humoral factors like antimicrobial peptides (AMPs) that kill the pathogens directly or is based on cellular processes involving phagocytosis and encapsulation by hemocytes. In Drosophila, the Toll pathway (activated by fungi and gram-positive bacteria) and the Imd pathway (activated by gram-negative bacteria) leads to the synthesis of AMPs. But AMP genes are also regulated without pathogenic challenge, e.g., by aging, circadian rhythms, and mating. This study shows that AMP genes are differentially expressed in mated females. Metchnikowin (Mtk) expression is strongly stimulated in the first 6 hr after mating. Sex-peptide (SP), a male seminal peptide transferred during copulation, is the major agent eliciting transcription of Mtk and of other AMP genes. Both pathways are needed for Mtk induction by SP. Furthermore, SP induces additional AMP genes via the Toll (Drosomycin) and the Imd (Diptericin) pathways. SP affects the Toll pathway at or upstream of the gene spätzle, and the Imd pathway at or upstream of the gene imd. Mating may physically damage females and pathogens may be transferred. Thus, endogenous stimulation of AMP transcription by SP at mating might be considered as a preventive step to encounter putative immunogenic attacks (Peng, 2005).

The Toll and Imd signaling cascades are the major and best-characterized pathways involved in the activation of AMPs after pathogenic challenges. The effect of SP on AMP expression was studied by comparing the expression of Mtk, Drs, and Dipt in wt females or in females mutant in the Toll and Imd pathways, respectively, before and after mating with wt males. RNA was extracted from virgin and mated females and analyzed by quantitative PCR (Peng, 2005).

With the exception of dorsal (dl), all loss-of-function mutants of the Toll and Imd pathways abolish or strongly reduce Mtk expression after mating. Thus, Mtk expression induced by SP is dependent on both pathways. Furthermore, since spz and imd females fail to induce Mtk transcription after mating, SP must act on or upstream of spz and imd. dl and its functional homolog dif have been reported to be involved in AMP gene transcription under pathogenic challenge in the larval stage, but not functional in the adult immune defense. A partial response is observed in dl females, indicating that dl may be partially involved in the innate immune response elicited by SP in adult females (Peng, 2005).

Drs expression, controlled by the Toll pathway, is completely abolished in spz and Tl mutants. Correspondingly, Dipt expression, which is controlled by the Imd pathway, is completely abolished in the Imd pathway loss-of-function mutants imd, Tak1, and rel. It is concluded that SP can activate the Toll and the Imd pathways. The Toll pathway is essential for Drs expression, whereas the Imd pathway is essential for Dipt expression (Peng, 2005).

The SP-induced immune response activates the transcription of all three AMP genes studied. After pathogenic infections, Drs is induced by the Toll pathway and Dipt by the Imd pathway, whereas both pathways induce Mtk expression. The results obtained with the loss-of-function mutants follow this scheme. Whereas loss-of-function mutants of both pathways reduce or abolish Mtk expression after mating, induction of Drs expression is only abolished by loss-of-function mutants of the Toll pathway, whereas induction of Dipt expression is only lost in mutants of the Imd pathway. In sum, the classical pathways are activated to induce the transcription of AMP genes after mating as after microbial or fungal infections (Peng, 2005).

Detection of microorganisms and triggering the appropriate pathway is achieved by pattern recognition receptors (PRRs), immune proteins recognizing general microbial components. Two families of PRRs have been identified in Drosophila: the peptidoglycan recognition proteins (PGRPs) and the gram-negative binding proteins (GNBPs). Some of the 13 PGRPs encoded in the D. melanogaster genome have been implicated in the activation of specific immune responses. However, the signaling cascades between the PRRs and the Toll and the Imd pathways are not well characterized. Since in spz and imd null mutants AMP induction by SP is specifically abolished, the inducing signals must affect the signaling cascades at or upstream of those genes. At this stage, it cannot be determined whether SP enters the pathways at the PRR level or at an intermediate level between the PRRs and spz or imd, respectively. Furthermore, the induction of AMPs may occur systemically (e.g., in the fat body) or locally in the reproductive tract. Microarray analysis of AMP expression after mating of wt females with either wt or SP⁰ males, respectively, suggests that AMPs are mainly induced in the abdomen, but it does not discriminate between a systematic response in the abdomen and a specific response in the genital tract (Peng, 2005).

Drosophila females undergo dramatic physiological changes after mating, predominantly induced by SP. Mating may also physically damage females and may expose the female to pathogens transferred by the male as shown for the milkweed leaf beetle. Thus, the activation of the innate immune system to encounter putative immunogenic attacks during this sensitive phase of the life history of females makes biological sense. The signal is plausibly coupled to copulation in the form of SP transferred in the seminal fluid. Such a mechanism might allow the female to respond preventively to potential threats. In sum, these findings may describe the result of an optimal economical balance between spending costly energy for the innate immune response and preventive measures to fight a putative pathogenic attack (Peng, 2005).

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date revised: 15 July 2014

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