Extracellular proteases and embryonic pattern formation.

At least three genes that play crucial roles in dorsal-ventral patterning of the Drosophila embryo appear to encode extracellular proteases. These proteases are involved in the generation of localized extracellular ligands for membrane receptors. Because the sequences of these gene products closely resemble those of mammalian enzymes that have been studied in detail biochemically, it is possible to draw on the wealth of information on the biochemical mechanisms that regulate protease activity to make inferences about how proteases can be used to generate spatial asymmetries within fields of cells.

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.

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.

Genetic characterization of tube and pelle, genes required for signaling between Toll and dorsal in the specification of the dorsal-ventral pattern of the Drosophila embryo.

tube and pelle are two of the maternally transcribed genes required for dorsal-ventral patterning of the Drosophila embryo. Females homozygous for strong alleles of tube or pelle produce embryos that lack all ventral and lateral embryonic pattern elements. By analyzing the phenotypes caused by 24 pelle and 9 tube alleles, we have defined characteristic features of the two genes, including the extremely variable phenotypes of a number of tube alleles and the antimorphic character of a number of pelle alleles. Double mutant females carrying dominant ventralizing alleles of Toll and dorsalizing alleles of tube or pelle produce dorsalized embryos, suggesting that tube and pelle act downstream of the membrane protein Toll in the signaling pathway that defines the embryonic dorsal-ventral pattern. Both tube and pelle are also important zygotically for survival: at least 30% of the zygotes lacking either tube or pelle die before adult stages, while 90-95% of tube- pelle- double mutant zygotes die. We discuss the phenotypes of tube-pelle double mutants in the context of whether the two proteins interact directly.

Zygotic expression and activity of the Drosophila Toll gene, a gene required maternally for embryonic dorsal-ventral pattern formation.

Maternal expression of the Toll gene is required for the production and the correct spatial organization of all lateral and ventral structures of the Drosophila embryo. We show here that the Toll gene is transcribed zygotically in the embryo and that zygotic expression is important for the viability of the larva. Both genetic and molecular data indicate that the zygotic Toll product has the same biochemical activity as the maternal product. The spatial distribution of the Toll transcript in the embryo was analyzed. In contrast to the uniform distribution of the maternal RNA, the zygotic Toll RNA is present in a complex spatial and temporal pattern in the embryo. A striking feature of this pattern is the correlation of the regions of invaginating cells with sites of accumulation of zygotic Toll RNA.

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.

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.

Neuronal receptive field properties in feline nucleus reticularis gigantocellularis.

Nucleus reticularis gigantocellularis (NGC) has been shown, using both behavioral and physiological techniques, to be involved in the processing of nociceptive information. However, previous studies of the receptive fields of NGC neurons have utilized only innocuous stimuli. Thus, while neurons in NGC may play an important role in nociception, the receptive field properties of these cells remain to be defined. This investigation was designed to determine the receptive field properties of neurons in NGC using nociceptive and innocuous stimuli. Receptive fields were determined for 127 neurons in NGC. Eighty-seven percnet of the NGC neurons studied responded exclusively to noxious stimuli, while 13% also responded to innocuous stimuli. None of the neurons studied responded exclusively to innocuous stimuli. The receptive fields of most NGC neurons (63%) were large, discontinuous, and bilaterally symmetrical. Eighty-one percent of NGC neurons received convergent inputs from both spinal and trigeminal systems. These receptive field properties differ from those previously reported using only innocuous stimulation.

Interactions between nucleus centrum medianum and gigantocellular nociceptive neurons.

Cells in nucleus reticularis gigantocellularis (NGC) and nucleus centrum medianum (CM) are known to respond to tooth pulp stimulation, which is a nociceptive trigeminal stimulus. We examined the effect of stimulation of CM on this class of neurons in NGC. Thirty-nine percent of the 57 neurons tested were antidromically activated by stimulation of the ipsilateral CM, while 28% of the 40 neurons tested were antidromically activated from the contralateral CM. In addition, the spontaneous activity of seven NGC neurons was altered by stimulation of the ipsilateral CM, while five cells were affected by stimulation of the contralateral CM. These data suggest a complex and reciprocal interaction between neurons in NGC and CM.

Response of cells in feline nucleus centrum medianum to tooth pulp stimulation.

Nucleus centrum medianum (CM) has been implicated in the processing of nociceptive information based on behavioral, physiological and clinical data. Although the responses of neurons in CM have been extensively studied using spinal stimuli, few data are available concerning their responses to trigeminal stimuli. This investigation was designed to determine the responses of cells in CM to tooth pulp stimulation, which has been demonstrated to be an effective and relatively specific trigeminal nociceptive stimulus. Responses were recorded from 378 neurons in 38 pentobarbital-anesthetized cats. Most cells responded at a latency of 5 to 40 msec with one to six spikes for each stimulus. Cells in the dorsal portion of CM tended to respond only to maxillary teeth, while cells in the ventral portion of the nucleus tended to respond only to mandibular teeth. Cells in the central portion of CM generally responded to both maxillary and mandibular teeth. Cells in CM generally responded to both ipsilateral and contralateral teeth, without a preference for one side. These data differ from those reported using spinal stimulation, since a topographic organization has not been identified with spinal stimulation.

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.

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.

Changing rates of histone mRNA synthesis and turnover in Drosophila embryos.

The rates of synthesis and turnover of histone mRNA in Drosophila embryos were determined by hybridization of in vivo and in vitro labeled embryonic RNA to Drosophila histone DNA of the recombinant plasmid cDm500. There is a large store of maternal histone mRNA, equivalent to at least 7 X 10(7) copies of each of the five classes of histone mRNA per embryo. Embryonic synthesis of histone mRNA begins at 90 min after oviposition, making the histone genes among the first to be transcribed by embryonic nuclei. Embryonic histone mRNA accumulates rapidly during the blastoderm and gastrula stages. The peak in the rate of histone mRNA synthesis per embryo coincides with the peak in the rate of DNA synthesis per embryo, which occurs at 6 hr after oviposition. After 6 hr, as the rate of DNA synthesis per embryo decreases, the rate of histone mRNA synthesis and the total mass of histone mRNA per embryo both drop sharply. The rate of histone mRNA synthesis per gene falls more than 60 fold in the first 13 hr after oviposition, from 1.3 -2.5 copies per gene-min at 2 hr to 0.02-0.03 copies per gene-min at 13 hr. From measurements of the mass of histone mRNA per embryo and of the rate of accumulation of newly synthesized histone mRNA at a number of stages of early embryogenesis we determined that the cytoplasmic half-life of histone mRNA decreases approximately 7 fold during early Drosophila development, from 2.3 hr at blastoderm to 20 min by the end of gastrulation. Thus the level of expression of histone genes in Drosophila development is controlled not only by the size of the maternal mRNA pool and changes in the rate of histone mRNA synthesis, but also by changes in the rate of histone mRNA turnover.

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.

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.

Decapentaplegic acts as a morphogen to organize dorsal-ventral pattern in the Drosophila embryo.

Zygotic expression of the Drosophila TGF beta family member decapentaplegic (dpp) is required for the development of the dorsal embryonic structures. By injecting dpp transcripts into young embryos, we find that 2- to 4-fold increases in the concentration of injected RNA elicit progressively more dorsal cell fates: only low levels of dpp permit development of ventral ectoderm, intermediate dpp levels drive dorsal epidermal development, and high dpp levels drive cells to differentiate as the most dorsal pattern element, the amnioserosa. Localized dpp RNA injections into embryos that lack all known maternal and zygotic dorsal-ventral polarity indicate that dpp can both define embryonic polarity and organize detailed patterning within the ectoderm. We infer that dpp acts as an extracellular morphogen and that the graded activity of dpp specifies the pattern of ectodermal cell fates in the Drosophila 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.

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.