Drosophila immune deficiency (IMD) is a death domain protein that activates antibacterial defense and can promote apoptosis.

We report the molecular characterization of the immune deficiency (imd) gene, which controls antibacterial defense in Drosophila. imd encodes a protein with a death domain similar to that of mammalian RIP (receptor interacting protein), a protein that plays a role in both NF-kappaB activation and apoptosis. We show that imd functions upstream of the DmIKK signalosome and the caspase DREDD in the control of antibacterial peptide genes. Strikingly, overexpression of imd leads to constitutive transcription of these genes and to apoptosis, and both effects are blocked by coexpression of the caspase inhibitor P35. We also show that imd is involved in the apoptotic response to UV irradiation. These data raise the possibility that antibacterial response and apoptosis share common control elements in Drosophila.

Constitutive activation of toll-mediated antifungal defense in serpin-deficient Drosophila.

The antifungal defense of Drosophila is controlled by the spaetzle/Toll/cactus gene cassette. Here, a loss-of-function mutation in the gene encoding a blood serine protease inhibitor, Spn43Ac, was shown to lead to constitutive expression of the antifungal peptide drosomycin, and this effect was mediated by the spaetzle and Toll gene products. Spaetzle was cleaved by proteolytic enzymes to its active ligand form shortly after immune challenge, and cleaved Spaetzle was constitutively present in Spn43Ac-deficient flies. Hence, Spn43Ac negatively regulates the Toll signaling pathway, and Toll does not function as a pattern recognition receptor in the Drosophila host defense.

Innate immunity in higher insects.

The hallmark of the innate immune response of higher insects is the rapid and transient synthesis of a battery of broad spectrum antimicrobial peptides by the fat body. The control of the genes encoding these peptides involves cis-regulatory promoter elements homologous to sequences functional in mammalian acute-phase genes. Study of immune-deficient mutants of Drosophila has indicated that distinct pathways control the antibacterial and antifungal responses in this species. Novel receptors potentially involved in the initiation of the immune response have been recently characterized.

The necrotic gene in Drosophila corresponds to one of a cluster of three serpin transcripts mapping at 43A1.2.

Mutants of the necrotic (nec) gene in Drosophila melanogaster die in the late pupal stage as pharate adults, or hatch as weak, but relatively normal-looking, flies. Adults develop black melanized spots on the body and leg joints, the abdomen swells with hemolymph, and flies die within 3 or 4 days of eclosion. The TOLL-mediated immune response to fungal infections is constitutively activated in nec mutants and pleiotropic phenotypes include melanization and cellular necrosis. These changes are consistent with activation of one or more proteolytic cascades. The nec gene corresponds to Spn43Ac, one of a cluster of three putative serine proteinase inhibitors at 43A1.2, on the right arm of chromosome 2. Although serpins have been implicated in the activation of many diverse pathways, lack of an individual serpin rarely causes a detectable phenotype. Absence of Spn43Ac, however, gives a clear phenotype, which will allow a mutational analysis of critical features of the molecular structure of serpins.

Dorsal-B, a splice variant of the Drosophila factor Dorsal, is a novel Rel/NF-kappaB transcriptional activator.

The Drosophila transcription factor Dorsal, a member of the Rel/NF-kappaB family of proteins, plays a key role in the establishment of dorsoventral polarity in the early embryo and is also involved in the immune response. Here, we present evidence that the primary transcript of dorsal can be alternatively spliced, generating Dorsal-B, a new Rel/NF-kappaB family member. Dorsal and Dorsal-B are identical in the N-terminal region, which comprises both a DNA-binding domain and a dimerization domain. However, Dorsal-B lacks the nuclear localization signal located at the end of the Rel domain of Dorsal and is totally divergent in the C-terminal portion. Although Dorsal-B by itself is not able to induce the expression of a kappaB-controlled Luciferase reporter gene, we demonstrate that its C-terminal portion has transactivating properties. Analysis of the dorsal-B expression pattern indicates that the splicing is tissue-specific and excludes a putative role in early embryogenesis. However, dorsal-B synthesis is enhanced upon septic injury, and this challenge induces a nuclear accumulation of the protein in fat body cells suggesting that it may be involved in the immune response.

The humoral antibacterial response of Drosophila.

Drosophila, like other insects, responds to the injection of bacteria by the rapid and transient synthesis of a battery of potent antibacterial peptides. Only a few of these peptides have been fully characterized to date. We review our recent data on the control of the expression of a gene encoding one of the induced peptides, i.e. diptericin. Our data highlight the role of proximal cis-regulatory motifs similar to regulatory elements binding NF-kappa B and NF-IL6 in promoters of some immune genes of mammals. We argue that the Drosophila host defense is homologous to the mammalian acute phase response.

Cg-Rel, the first Rel/NF-kappaB homolog characterized in a mollusk, the Pacific oyster Crassostrea gigas.

We report here the identification and functional characterization of Cg-Rel, a gene encoding the Crassostrea gigas homolog of Rel/NF-kappaB transcription factors found in insects and mammals. Sequence and phylogenetic analysis showed that Cg-Rel shares the structural organization of Rel/NF-kappaB transcription factors of class II. It includes a Rel homology domain as well as a C-terminal transactivation domain (TD). Overexpression of Cg-Rel in the Drosophila S2 cell line activated the expression of a NF-kappaB-dependent reporter gene, whereas transfection with a Cg-Rel construct containing a C-terminal deletion of the TD or using a reporter gene with mutated kappaB binding sites failed to activate expression. These results suggest that Cg-Rel is a functional member of the Rel family of transcription factors, making this the sixth structurally homologous component of the Rel/NF-kappaB pathway characterized in C. gigas. Based on homology to other invertebrates' Rel/NF-kappaB cascade, the function of the oyster pathway may serve to regulate genes involved in innate defense and/or development. These findings serve to highlight a potentially important regulatory pathway to the study of oyster immunology, hence allowing comparison of the immune system in vertebrates and invertebrates, an important key issue to understand its evolution.

Determination of the disulfide array of the first inducible antifungal peptide from insects: drosomycin from Drosophila melanogaster.

Drosomycin is a 44-residue antifungal peptide with four intramolecular disulfide bridges which have been isolated from immune-challenged Drosophila. To produce adequate amounts of this peptide for 3D-structure analysis, studies on the mode of action and activity spectrum, we expressed a synthetic cDNA in Saccharomyces cerevisiae. For this purpose, we used the mating factor alpha gene and concomitantly overexpressed the KEX2 gene to increase the yield of fully processed drosomycin. Using a combination of Edman degradation and mass spectrometry, we show that drosomycin shares the same array of intramolecular disulfide bridges than plant defensins, in addition to their sequence similarities.

Pharmacological characteristics and anatomical distribution of [3H]oxytocin-binding sites in the Wistar rat brain studied by autoradiography.

Oxytocin-binding sites were detected by autoradiography on rat brain sections incubated in the presence of the [3H]oxytocin. These sites were characterized pharmacologically using quantitative autoradiography. High pressure liquid chromatography controls of the incubation media indicated that labelling was due to the intact [3H]oxytocin molecule. Pharmacological analysis of different locations (central amygdaloid nucleus, ventral subiculum and ventromedial hypothalamic nucleus) showed that the sites detected had a high affinity for oxytocin and also for arginine-vasopressin. In contrast, some areas known to bind vasopressin intensely, such as suprachiasmatic and lateral septum nuclei, had little or no affinity for oxytocin. Autoradiographs revealed [3H]oxytocin-binding sites in already known brain areas (olfactory centres, ventral subiculum, central amygdaloid nucleus, bed nucleus of the stria terminalis) albeit with more extensive labelling of some of these formations, in particular, the amygdaloid complex. In addition, specific [3H]oxytocin-binding sites were found in areas not yet reported to bind oxytocin, such as the paraventricular thalamic and caudate nuclei. In the hypothalamus, specific binding sites were not detected in the supraoptic and paraventricular nuclei: the only structure labelled was the ventrolateral part of the ventromedial nucleus. Discrepancies between the concentrations of [3H]oxytocin-binding sites, the known distribution of oxytocin-containing endings and electrophysiological data indicate that autoradiography, under our conditions, apparently only reveals some of the oxytocin receptors in the brain. Thus, in the hypothalamus, no relationship can be established between the known effect of oxytocin on oxytocinergic magnocellular neurons and detection of specific [3H]oxytocin-binding sites. Autoradiography may reveal mainly oxytocin-binding sites in areas receiving diverse "parasynaptic" information, where oxytocin might play a modulatory role rather than exerting rapid, short-term effects of the neurotransmitter type.

[Current findings on the asymmetric growth of the mullerian tract in female chick embryos]. / Nouvelles données sur la croissance asymétrique du tractus müllérien chez l'embryon de poulet femelle.

Shape and orientation of the mesothelial cells were examined in the mullerian ducts of 8, 13 and 15 day female chick embryos with the scanning electron microscope. The observed evolution in the pattern of these cells likely reflects the mechanical conditions to which these organs are subjected during embryonic development: stretching for the left duct, slackening for the right duct. These observations, together with data concerning growth of these organs, suggest that topographical relationships between cells, which in this system result from mechanical factors, play an important role in controlling cell proliferation.

Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms.

Insects respond to microbial infection by the rapid and transient expression of several genes encoding potent antimicrobial peptides. Herein we demonstrate that this antimicrobial response of Drosophila is not aspecific but can discriminate between various classes of microorganisms. We first observe that the genes encoding antibacterial and antifungal peptides are differentially expressed after injection of distinct microorganisms. More strikingly, Drosophila that are naturally infected by entomopathogenic fungi exhibit an adapted response by producing only peptides with antifungal activities. This response is mediated through the selective activation of the Toll pathway.

Growth stimulation of the immature chick oviduct by androgens: the vagina as a new target tissue.

Testosterone when injected alone stimulates growth of the vagina but is inactive upon the other segments of the oviduct of the immature chicken. This action of testosterone can already be detected in embryos: it is expressed by the beginning of differentiation of the vaginal mesenchyme cells into smooth muscle cells. In the treated immature chicken, stimulation of growth is considerable and is specifically caused by androgens (testosterone and 5-alpha-dihydrotestosterone); the vaginal mesenchyme differentiates into two smooth muscular layers and vaginal epithelium cells differentiate into ciliated cells and goblets cells. [3H]testosterone binding has been found in the vagina of the immature chicken (data not shown). The characteristics of testosterone binding to cytoplasmic components of the chick vagina are consistent with its identity as a testosterone receptor.

L-canavanine incorporation into vitellogenin and macromolecular conformation.

L-Canavanine is a potentially deleterious arginine antimetabolite whose toxicity is expressed in canavanine-sensitive organisms ranging from viruses to humans. Canavanine, a substrate for arginyl-tRNA synthetase, is incorporated into nascent polypeptide chains in place of arginine. This substitution results in the production of structurally aberrant, canavanyl proteins. Chemical, physical, and immunological studies of native and canavanine-containing vitellogenin obtained from female migratory locusts (Locusta migratoria migratorioides (Orthoptera] provide the first experimental evidence that canavanine can disrupt the tertiary and/or quaternary structure that yields the three-dimensional conformation unique to the protein. These findings enhance our understanding of the biochemical basis for canavanine's antimetabolic and potent insecticidal properties.

Insect immunity. A transgenic analysis in Drosophila defines several functional domains in the diptericin promoter.

Diptericins are antibacterial polypeptides which are strongly induced in the fat body and blood cells of dipteran insects in response to septic injury. The promoter of the single-copy, intronless diptericin gene of Drosophila contains several nucleotide sequences homologous to mammalian cis-regulatory motifs involved in the control of acute phase response genes. Extending our previous studies on the expression of the diptericin gene, we now report a quantitative analysis of the contribution of various putative regulatory elements to the bacterial inducibility of this gene, based on the generation of 60 transgenic fly lines carrying different elements fused to a reporter gene. Our data definitively identify two Kappa B-related motifs in the proximal promoter as the sites conferring inducibility and tissue-specific expression to the diptericin gene. These motifs alone, however, mediate only minimal levels of expression. Additional proximal regulatory elements are necessary to attain some 20% of the full response and we suspect a role for sequences homologous to mammalian IL6 response elements and interferon-gamma responsive sites in this up-regulation. The transgenic experiments also reveal the existence of a distal regulatory element located upstream of -0.6 kb which increases the level of expression by a factor of five.

Expression and nuclear translocation of the rel/NF-kappa B-related morphogen dorsal during the immune response of Drosophila.

The rel/NF-kappa B-related morphogen dorsal is a maternally expressed gene which is involved in the control of the dorso-ventral axis during early embryogenesis of Drosophila. We show that this gene is also expressed in the fat body of larvae and adults of Drosophila as well as in a tumorous blood cell line: its expression is noticeably enhanced upon bacterial (or lipopolysaccharide) challenge. This challenge also induces within 15-30 min a nuclear translocation of the dorsal protein. The genes encoding inducible antibacterial peptides in Drosophila contain kappa B-related nucleotide sequences and we show that the dorsal protein can bind to such motifs and sequence-specifically transactivate a reporter gene in co-transfection experiments with a Drosophila cell line. However, in dl1 mutants, in the absence of dorsal protein, the genes encoding antibacterial peptides retain their inducibility, suggesting a multifactorial control. The results indicate that in addition to its role in embryogenesis, dorsal is involved in the immune response of Drosophila. They also strengthen the analogy between the mammalian acute phase response and the insect immune response.

Insect immunity. Two 17 bp repeats nesting a kappa B-related sequence confer inducibility to the diptericin gene and bind a polypeptide in bacteria-challenged Drosophila.

The Drosophila diptericin gene codes for a 9 kDa antibacterial peptide and is rapidly and transiently expressed in larvae and adults after bacterial challenge. It is also induced in a tumorous Drosophila blood cell line by the addition of lipopolysaccharide (LPS). The promoter of this gene contains two 17 bp repeats located closely upstream of the TATA-box and harbouring a decameric kappa B-related sequence. This study reports that the replacement of the two 17 bp repeats by random sequences abolishes bacteria inducibility in transgenic fly lines. In transfected tumorous blood cells, the replacement of both or either of the 17 bp motifs reduces dramatically LPS inducibility, whereas multiple copies significantly increase the level of transcriptional activation by LPS challenge. A specific DNA-protein binding activity is evidenced in cytoplasmic and nuclear extracts of induced blood cells and fat body. It is absent in controls. It is proposed that induction of the diptericin gene mediated by the two 17 bp repeats occurs via a mechanism similar to that of mammalian NF-kappa B.

Insect immunity: the diptericin promoter contains multiple functional regulatory sequences homologous to mammalian acute-phase response elements.

We are using the diptericin gene as a model system to study the control of expression of the genes encoding antibacterial peptides during the Drosophila immune reaction. In order to investigate the putative regulatory regions in the diptericin promoter, we performed DNaseI footprinting experiments combined with gel-shift assays in two inducible systems: the larval fat body and a tumorous Drosophila blood cell line. Our results confirm the importance of kappa B-like elements previously described in the immune response of insects and reveal for the first time the involvement of other regions containing sequences homologous to mammalian acute-phase response elements.

Cloning of the gene encoding the antibacterial peptide drosocin involved in Drosophila immunity. Expression studies during the immune response.

A potent inducible antibacterial peptide carrying an O-glycosylated substitution has recently been isolated from Drosophila [Bulet, P., Dimarcq, J. L., Hetru, C., Lagueux, M., Charlet, M., Hegy, G., Van Dorsselaer, A. and Hoffmann, J. A. (1993) J. Biol. Chem. 268, 14893-14897]. Here we report cloning studies that show that Drosophila contains a single, intronless gene, located at position 51C1-6, which encodes the precursor protein from which drosocin is processed. The upstream and the downstream sequences of the drosocin gene contain putative cis-regulatory elements similar to mammalian regulatory motifs, namely three kappa B-related decameric sequences. The drosocin gene is silent in naive animals, and is strongly induced with acute phase kinetics after immune challenge in larvae and in adults. We have established several transgenic fly lines in which reporter genes were placed under the control of various drosocin promoter sequences. Our results indicate that 2.5 kb of upstream sequences confer inducibility and tissue specificity to the transgene, but that the level of its expression in the fat body after immune challenge is low. Addition of genomic regions downstream of the drosocin transcribed sequences results in increased transcription levels, which are similar for the fusion and the resident drosocin genes upon infection. Analysis of transgenic fly lines showed that the drosocin reporter gene is constitutively expressed in the oviducts of egg-laying females.

The dorsoventral regulatory gene cassette spätzle/Toll/cactus controls the potent antifungal response in Drosophila adults.

The cytokine-induced activation cascade of NF-kappaB in mammals and the activation of the morphogen dorsal in Drosophila embryos show striking structural and functional similarities (Toll/IL-1, Cactus/I-kappaB, and dorsal/NF-kappaB). Here we demonstrate that these parallels extend to the immune response of Drosophila. In particular, the intracellular components of the dorsoventral signaling pathway (except for dorsal) and the extracellular Toll ligand, spätzle, control expression of the antifungal peptide gene drosomycin in adults. We also show that mutations in the Toll signaling pathway dramatically reduce survival after fungal infection. Antibacterial genes are induced either by a distinct pathway involving the immune deficiency gene (imd) or by combined activation of both imd and dorsoventral pathways.

A mosaic analysis in Drosophila fat body cells of the control of antimicrobial peptide genes by the Rel proteins Dorsal and DIF.

Expression of the gene encoding the antifungal peptide Drosomycin in Drosophila adults is controlled by the Toll signaling pathway. The Rel proteins Dorsal and DIF (Dorsal-related immunity factor) are possible candidates for the transactivating protein in the Toll pathway that directly regulates the drosomycin gene. We have examined the requirement of Dorsal and DIF for drosomycin expression in larval fat body cells, the predominant immune-responsive tissue, using the yeast site-specific flp/FRT recombination system to generate cell clones homozygous for a deficiency uncovering both the dorsal and the dif genes. Here we show that in the absence of both genes, the immune-inducibility of drosomycin is lost but can be rescued by overexpression of either dorsal or dif under the control of a heat-shock promoter. This result suggests a functional redundancy between both Rel proteins in the control of drosomycin gene expression in the larvae of Drosophila. Interestingly, the gene encoding the antibacterial peptide Diptericin remains fully inducible in the absence of the dorsal and dif genes. Finally, we have used fat body cell clones homozygous for various mutations to show that a linear activation cascade Spaetzle--> Toll-->Cactus-->Dorsal/DIF leads to the induction of the drosomycin gene in larval fat body cells.