Drosophila immunity: genes on the third chromosome required for the response to bacterial infection.

We have screened the third chromosome of Drosophila melanogaster for mutations that prevent the normal immune response. We identified mutant lines on the basis of their failure to induce transcription of an antibacterial peptide gene in response to infection or their failure to form melanized clots at the site of wounding. These mutations define 14 genes [immune response deficient (ird) genes] that have distinct roles in the immune response. We have identified the molecular basis of several ird phenotypes. Two genes, scribble and kurtz/modulo, affect the cellular organization of the fat body, the tissue responsible for antimicrobial peptide production. Two ird genes encode components of the signaling pathways that mediate responses to bacterial infection, a Drosophila gene encoding a homolog of I kappa B kinase (DmIkk beta) and Relish, a Rel-family transcription factor. These genetic studies should provide a basis for a comprehensive understanding of the genetic control of immune responses in Drosophila.

Rab23 is an essential negative regulator of the mouse Sonic hedgehog signalling pathway.

The mouse open brain (opb) and Sonic hedgehog (Shh) genes have opposing roles in neural patterning: opb is required for dorsal cell types and Shh is required for ventral cell types in the spinal cord. Here we show that opb acts downstream of Shh. Ventral cell types that are absent in Shh mutants, including the floor plate, are present in Shh opb double mutants. The organization of ventral cell types in Shh opb double mutants reveals that Shh-independent mechanisms can pattern the neural tube along its dorsal-ventral axis. We cloned opb by a map-based approach and found that it encodes Rab23, a member of the Rab family of vesicle transport proteins. The data indicate that dorsalizing signals activate transcription of Rab23 in order to silence the Shh pathway in dorsal neural cells.

fusilli, an essential gene with a maternal role in Drosophila embryonic dorsal-ventral patterning.

The Drosophila fusilli (fus) gene was identified in a genetic screen for dominant maternal enhancers of an unusual dorsalizing mutation in the cactus gene, cact(E10). While females that are heterozygous for the cact(E10) allele produce embryos with wild-type dorsal-ventral patterning, more than 90% of the embryos produced by females that are heterozygous for both cact(E10) and the fus(1) mutation are weakly dorsalized. Loss of fusilli activity causes lethality during embryogenesis but not dorsal-ventral patterning defects, indicating that fusilli is important in more than one developmental process. The fusilli gene encodes a protein with RNA binding motifs related to those in mammalian hnRNP F and H, which play roles in regulated RNA splicing. The fusilli RNA is not present in the oocyte or early embryo, and germ-line clones of fusilli mutations have no maternal effect on dorsal-ventral patterning, indicating that the fusilli maternal effect does not depend on germ-line expression of the gene. Because the fusilli RNA is present in ovarian follicle cells, we propose that fusilli acts downstream of the Drosophila EGF receptor to control the biogenesis of follicle cell transcripts that control the initial dorsal-ventral asymmetry of the embryo.

The antibacterial arm of the drosophila innate immune response requires an IkappaB kinase.

The ird5 gene was identified in a genetic screen for Drosophila immune response mutants. Mutations in ird5 prevent induction of six antibacterial peptide genes in response to infection but do not affect the induction of an antifungal peptide gene. Consistent with this finding, Escherichia coli survive 100 times better in ird5 adults than in wild-type animals. The ird5 gene encodes a Drosophila homolog of mammalian IkappaB kinases (IKKs). The ird5 phenotype and sequence suggest that the gene is specifically required for the activation of Relish, a Drosophila NF-kappaB family member.

Dorsal and lateral fates in the mouse neural tube require the cell-autonomous activity of the open brain gene.

The processes that specify early regional identity in dorsal and lateral regions of the mammalian neural tube are not well understood. The mouse open brain (opb) gene plays an essential role in dorsal neural patterning: in the caudal spinal cord of opb mutants, dorsal cell types are absent and markers of ventral fates, including Shh, expand into dorsal regions. Analysis of the opb mutant phenotype and of opb/opb <--> wild-type chimeric embryos reveals that early in neural development, the wild-type opb gene (opb(+)) is required cell autonomously for the expression of Pax7 in dorsal cells and Pax6 in lateral cells. Thus the opb(+) gene product acts intracellularly in the reception or interpretation of signals that determine cell types in the dorsal 80% of the neural tube. At later stages, the lack of opb(+) causes a non-cell-autonomous expansion of ventral cell types into dorsal regions of the neural tube, revealing that opb(+) controls the production of a diffusible molecule that defines the domain of Shh expression. The data indicate that opb(+) could act as either a novel component of a dorsalizing pathway or a novel intracellular negative regulator of the Shh signal transduction pathway.

Toll signaling pathways in the innate immune response.

The Toll signaling pathway, which is required for the establishment of the dorsal-ventral axis in Drosophila embryos, plays an important role in the response of larval and adult Drosophila to microbial infections. Recent genetic evidence has shown that a mammalian Toll-like receptor, mouse Tlr4, is the signal transducing receptor activated by bacterial lipopolysaccharide. Thus, Toll-like receptors appear to detect a variety of microbial components and to trigger a defensive reaction in both Drosophila and mammals. Genetic data from both Drosophila and mice have defined components required for activation of Toll-like receptors and for the downstream pathways activated by the Toll-like receptors.

Finding the genes that direct mammalian development : ENU mutagenesis in the mouse.

The genetic control of mammalian embryogenesis is not well understood. N-ethyl-N-nitrosourea (ENU) mutagenesis screens in the mouse provide a route to identify more of the genes that are required for mammalian development. The characterization of ENU-induced mutations can build on the resources provided by the mouse and human genome projects to help define the tissue interactions and signaling pathways that direct early mammalian development.

Altered patterns of gene expression in Tribolium segmentation mutants.

Embryos homozygous for the godzilla, jaws, or kraken mutations of Tribolium castaneum have characteristic defects in segmentation. Here, we examine the expression of Tribolium genes homologous to Drosophila segmentation genes in these mutants to define similarities and differences in the process of segmentation in the two insects. The godzilla mutation disrupts segmentation and alters the expression of Even-skipped (Eve) throughout the germ band. godzilla, therefore, acts at an early step in the segmentation gene hierarchy; the evidence suggests it could be the Tribolium homologue of the eve gene. The jaws mutation causes deletion of most of the abdomen; this defect appears to be correlated with the failure of Eve to resolve into a striped pattern of expression, jaws also causes homeotic transformations in the thorax and the first abdominal segment; this transformation is accompanied by ectopic expression of the homeotic gene maxillopedia in the transformed segments. The kraken mutant disrupts patterning within each segment and, like Drosophila segment polarity mutants, disrupts the maintenance of the normal expression domain of Engrailed. This analysis suggests that late stages of segmentation are similar in Tribolium and Drosophila, although there are clear differences in early steps of segmentation in the two insects.

A phenotype-based screen for embryonic lethal mutations in the mouse.

The genetic pathways that control development of the early mammalian embryo have remained poorly understood, in part because the systematic mutant screens that have been so successful in the identification of genes and pathways that direct embryonic development in Drosophila, Caenorhabditis elegans, and zebrafish have not been applied to mammalian embryogenesis. Here we demonstrate that chemical mutagenesis with ethylnitrosourea can be combined with the resources of mouse genomics to identify new genes that are essential for mammalian embryogenesis. A pilot screen for abnormal morphological phenotypes of midgestation embryos identified five mutant lines; the phenotypes of four of the lines are caused by recessive traits that map to single regions of the genome. Three mutant lines display defects in neural tube closure: one is caused by an allele of the open brain (opb) locus, one defines a previously unknown locus, and one has a complex genetic basis. Two mutations produce novel early phenotypes and map to regions of the genome not previously implicated in embryonic patterning.

Positive and negative regulation of Easter, a member of the serine protease family that controls dorsal-ventral patterning in the Drosophila embryo.

The sequential activities of four members of the trypsin family of extracellular serine proteases are required for the production of the ventrally localized ligand that organizes the dorsal-ventral pattern of the Drosophila embryo. The last protease in this sequence is encoded by easter, which is a candidate to activate proteolytically the ligand encoded by spätzle. Here, we demonstrate biochemically that the zymogen form of Easter is processed in vivo by a proteolytic cleavage event that requires the three upstream proteases. Processed Easter is present in extremely low amounts in the early embryo because it is rapidly converted into a high molecular mass complex, which may contain a protease inhibitor. Easter zymogen activation is also controlled by a negative feedback loop from Dorsal, the transcription factor at the end of the signaling pathway. Each of these regulated biochemical processes is likely to be important in generating the ventral-to-dorsal gradient of Dorsal protein that organizes cell fates in the early embryo.

Regulated nuclear import of Rel proteins in the Drosophila immune response.

The Drosophila immune response uses many of the same components as the mammalian innate immune response, including signalling pathways that activate transcription factors of the Rel/NK-kappaB family. In response to infection, two Rel proteins, Dif and Dorsal, translocate from the cytoplasm to the nuclei of larval fat-body cells. The Toll signalling pathway, which controls dorsal-ventral patterning during Drosophila embryogenesis, regulates the nuclear import of Dorsal in the immune response, but here we show that the Toll pathway is not required for nuclear import of Dif. Cytoplasmic retention of both Dorsal and Dif depends on Cactus protein; nuclear import of Dorsal and Dif is accompanied by degradation of Cactus. Therefore the two signalling pathways that target Cactus for degradation must discriminate between Cactus-Dorsal and Cactus-Dif complexes. We identified new genes that are required for normal induction of transcription of an antibacterial peptide during the immune response. Mutations in three of these genes prevent nuclear import of Dif in response to infection, and define new components of signalling pathways involving Rel. Mutations in three other genes cause constitutive nuclear localization of Dif; these mutations may block Rel protein activity by a novel mechanism.

Embryonic patterning mutants of Tribolium castaneum.

The identification and analysis of genes controlling segmentation in Drosophila melanogaster has opened the way for understanding similarities and differences in mechanisms of segmentation among the insects. Homologues of Drosophila segmentation genes have been cloned and their expression patterns have been analyzed in a variety of insects, revealing that the patterns of expression of many genes are conserved. Conserved expression patterns do not, however, necessarily reflect conserved gene function. To address gene function, we have conducted a screen for mutations that alter embryonic patterning of the beetle, Tribolium castaneum. One of the mutations isolated, godzilla, affects early steps in the segmentation process in the whole animal, like Drosophila pair-rule mutants. Another mutation, jaws, is novel: it caused both a dramatic homeotic transformation in the thorax and first abdominal segment as well as a deletion of most of the segments of the abdomen. In Tribolium and other intermediated germ band insects, the anterior segments of the embryo are determined in the syncytium of the blastoderm, whereas the abdominal segments proliferated in the cellular environment. Both the godzilla and jaws mutations affect segments that are formed in the syncytium differently from those that are formed after cellularization. These regionally specific phenotypes may reflect the different patterning mechanisms that must be employed by the anterior and posterior regions of an intermediated germ insect.

A conserved signaling pathway: the Drosophila toll-dorsal pathway.

The Toll-Dorsal pathway in Drosophila and the interleukin-1 receptor (IL-1R)-NF-kappa B pathway in mammals are homologous signal transduction pathways that mediate several different biological responses. In Drosophila, genetic analysis of dorsal-ventral patterning of the embryo has defined the series of genes that mediate the Toll-Dorsal pathway. Binding of extracellular ligand activates the transmembrane receptor Toll, which requires the novel protein Tube to activate the cytoplasmic serine/threonine kinase Pelle. Pelle activity controls the degradation of the Cactus protein, which is present in a cytoplasmic complex with the Dorsal protein. Once Cactus is degraded in response to signal, Dorsal is free to move into the nucleus where it regulates transcription of specific target genes. The Toll, tube, pelle, cactus, and dorsal genes also appear to be involved in Drosophila immune response. Because the IL-1R-NF-kappa B pathway plays a role in vertebrate innate immunity and because plant homologues of the Toll-Dorsal pathway are important in plant disease resistance, it is likely that this pathway arose before the divergence of plants and animals as a defense against pathogens.

Changes in plasma triacylglycerol concentrations among free-living hyperlipidemic men adopting different carbohydrate intakes over 2 y: the Dietary Alternatives Study.

We reported previously that low-fat, high-carbohydrate diets containing < 26% of energy as fat and > 57% of energy as carbohydrate induce hypertriacylglycerolemia (hypertriglyceridemia) in hypercholesterolemic but not in combined hyperlipidemic (CHL) subjects. Because subjects may not consistently adhere to an assigned diet long term, we examined the extent to which plasma triacylglycerols (triglycerides) increase at four consistently reported carbohydrate intakes at intervals of up to 2 y. Three hundred seventy-two subjects reported consistent carbohydrate intakes of < 45%, 45-51.9%, 52-59.9%, or > or = 60% of energy on food records for 3, 12, and 24 mo. Among hypercholesterolemic subjects reporting a carbohydrate intake > or = 60% of energy, triacylglycerols increased by 0.25, 0.18, and 0.27 mmol/L (22, 16, and 24 mg/dL) over baseline at 3, 12, and 24 mo, respectively (P < 0.01 in each instance), and 0.32 mmol/L (28 mg/dL) above the group with a carbohydrate intake 52-59.9% of energy (P < 0.05) after 3 mo. No statistically significant effects were observed among CHL subjects, but compared with baseline, triacylglycerols decreased during the first 3 mo (-0.29 to -0.04 mmol/L, or -26 to -4 mg/dL), were unchanged over 12 mo, and were increased after 24 mo in three of four carbohydrate intake strata (0.27-0.36 mmol/L, or 24-32 mg/dL). These data confirm our previous observation that a moderately but not extremely low-fat, high-carbohydrate diet can be used long-term without deleterious effects on plasma triacylglycerols in the management of hypercholesterolemia, whereas CHL is unaffected by the amount of carbohydrate ingested.

Cactus protein degradation mediates Drosophila dorsal-ventral signaling.

Dorsal-ventral patterning in the Drosophila embryo relies on a signal transduction pathway that is similar to a signaling pathway leading to the activation of the mammalian transcription factor NF-kappa B. Stimulation of this Drosophila pathway on the ventral side of the embryo causes the nuclear translocation of Dorsal, the Drosophila NF-kappa B homolog. Cactus, like its mammalian homolog I kappa B, inhibits nuclear translocation by binding Dorsal and retaining it in the cytoplasm. We show that Cactus, like I kappa B, is rapidly degraded in response to signaling. More importantly, signal-dependent degradation of Cactus does not require the presence of Dorsal, indicating that Cactus degradation is a direct response to signaling, and that disruption of the Dorsal/Cactus complex is a secondary result of Cactus degradation. Mutant alleles of cactus that encode more stable forms of the protein block signaling, showing that efficient degradation is necessary for signaling. We find that Cactus protein stability is regulated by two independent processes that rely on different regions within the protein: signal-dependent degradation requires sequences in the amino terminus or ankyrin repeats, whereas signal-independent degradation of free Cactus requires the carboxy-terminal region of the protein that includes a PEST sequence.

Signaling pathways that establish the dorsal-ventral pattern of the Drosophila embryo.

The dorsal-ventral pattern of the Drosophila embryo is established by three sequential signaling pathways. Each pathway transmits spatial information by localizing the activity of an extracellular signal, which acts as a ligand for a broadly distributed transmembrane receptor. The components of the first two pathways are encoded by maternal effect genes, while the third pathway is specified by genes expressed in the zygote. During oogenesis, the oocyte transmits a signal to the surrounding follicle cells by the gurken-torpedo pathway. After fertilization, the initial asymmetry of the egg chamber is used by the spätzle-Toll pathway to generate within the embryo a nuclear gradient of the transcription factor Dorsal, which regulates the regional expression of a set of zygotic genes. On the dorsal side of the embryo, the decapentaplegic-punt/thick veins pathway then establishes patterning of the amnioserosa and dorsal ectoderm. Each pathway uses a distinct strategy to achieve spatial localization of signaling activity.

A processed form of the Spätzle protein defines dorsal-ventral polarity in the Drosophila embryo.

Stein et al. (1991) identified a soluble, extracellular factor that induces ventral structures at the site where it is injected in the extracellular space of the early Drosophila embryo. This factor, called polarizing activity, has the properties predicted for a ligand for the transmembrane receptor encoded by the Toll gene. Using a bioassay to follow activity, we purified a 24 x 10(3) M(r) protein that has polarizing activity. The purified protein is recognized by antibodies to the C-terminal half of the Spätzle protein, indicating that this polarizing activity is a product of the spätzle gene. The purified protein is smaller than the primary translation product of spätzle, suggesting that proteolytic processing of Spätzle on the ventral side of the embryo is required to generate the localized, active form of the protein.

The spätzle gene encodes a component of the extracellular signaling pathway establishing the dorsal-ventral pattern of the Drosophila embryo.

spätzle is a maternal effect gene required in the signal transduction pathway that establishes the dorsal-ventral pattern of the Drosophila embryo. spätzle acts immediately upstream of the membrane protein Toll in the genetic pathway, suggesting that spätzle could encode the ventrally localized ligand that activates the receptor activity of Toll. The spätzle gene encodes a novel secreted protein that appears to require activation by a proteolytic processing reaction, which is controlled by the genes that act upstream of spätzle in the genetic pathway. We propose that proteolytic processing of the spätzle protein is confined to the ventral side of the embryo and that the localization of processed spätzle determines where the receptor, Toll, is active.