High sugar-induced insulin resistance in Drosophila relies on the lipocalin Neural Lazarillo

In multicellular organisms, insulin/IGF signaling (IIS) plays a central role in matching energy needs with uptake and storage, participating in functions as diverse as metabolic homeostasis, growth, reproduction and ageing. In mammals, this pleiotropy of action relies in part on a dichotomy of action of insulin, IGF-I and their respective membrane-bound receptors. In organisms with simpler IIS, this functional separation is questionable. In Drosophila IIS consists of several insulin-like peptides called Dilps, activating a unique membrane receptor and its downstream signaling cascade. During larval development, IIS is involved in metabolic homeostasis and growth. We have used feeding conditions (high sugar diet, HSD) that induce an important change in metabolic homeostasis to monitor possible effects on growth. Unexpectedly we observed that HSD-fed animals exhibited severe growth inhibition as a consequence of peripheral Dilp resistance. Dilp-resistant animals present several metabolic disorders similar to those observed in type II diabetes (T2D) patients. By exploring the molecular mechanisms involved in Drosophila Dilp resistance, we found a major role for the lipocalin Neural Lazarillo (NLaz), a target of JNK signaling. NLaz expression is strongly increased upon HSD and animals heterozygous for an NLaz null mutation are fully protected from HSD-induced Dilp resistance. NLaz is a secreted protein homologous to the Retinol-Binding Protein 4 involved in the onset of T2D in human and mice. These results indicate that insulin resistance shares common molecular mechanisms in flies and human and that Drosophila could emerge as a powerful genetic system to study some aspects of this complex syndrome.     

Grasshopper hunchback expression reveals conserved and novel aspects of axis formation and segmentation

While the expression patterns of segment polarity genes such as engrailed have been shown to be similar in Drosophila melanogaster and Schistocerca americana (grasshopper), the expression patterns of pair-rule genes such as even-skipped are not conserved between these species. This might suggest that the factors upstream of pair-rule gene expression are not conserved across insect species. We find that, despite this, many aspects of the expression of the Drosophila gap gene hunchback are shared with its orthologs in the grasshoppers S. americana and L. migratoria. We have analyzed both mRNA and protein expression during development, and find that the grasshopper hunchback orthologs appear to have a conserved role in early axial patterning of the germ anlagen and in the specification of gnathal and thoracic primordia. In addition, distinct stepped expression levels of hunchback in the gnathal/thoracic domains suggest that grasshopper hunchback may act in a concentration-dependent fashion (as in Drosophila), although morphogenetic activity is not set up by diffusion to form a smooth gradient. Axial patterning functions appear to be performed entirely by zygotic hunchback, a fundamental difference from Drosophila in which maternal and zygotic hunchback play redundant roles. In grasshoppers, maternal hunchback activity is provided uniformly to the embryo as protein and, we suggest, serves a distinct role in distinguishing embryonic from extra-embryonic cells along the anteroposterior axis from the outset of development - a distinction made in Drosophila along the dorsoventral axis later in development. Later hunchback expression in the abdominal segments is conserved, as are patterns in the nervous system, and in both Drosophila and grasshopper, hunchback is expressed in a subset of extra-embryonic cells. Thus, while the expected domains of hunchback expression are conserved in Schistocerca, we have found surprising and fundamental differences in axial patterning, and have identified a previously unreported domain of expression in Drosophila that suggests conservation of a function in extra-embryonic patterning.     

The peripheral nervous system supports blood cell homing and survival in the Drosophila larva

Interactions of hematopoietic cells with their microenvironment control blood cell colonization, homing and hematopoiesis. Here, we introduce larval hematopoiesis as the first Drosophila model for hematopoietic colonization and the role of the peripheral nervous system (PNS) as a microenvironment in hematopoiesis. The Drosophila larval hematopoietic system is founded by differentiated hemocytes of the embryo, which colonize segmentally repeated epidermal-muscular pockets and proliferate in these locations. Importantly, we show that these resident hemocytes tightly colocalize with peripheral neurons and we demonstrate that larval hemocytes depend on the PNS as an attractive and trophic microenvironment. atonal (ato) mutant or genetically ablated larvae, which are deficient for subsets of peripheral neurons, show a progressive apoptotic decline in hemocytes and an incomplete resident hemocyte pattern, whereas supernumerary peripheral neurons induced by ectopic expression of the proneural gene scute (sc) misdirect hemocytes to these ectopic locations. This PNS-hematopoietic connection in Drosophila parallels the emerging role of the PNS in hematopoiesis and immune functions in vertebrates, and provides the basis for the systematic genetic dissection of the PNS-hematopoietic axis in the future.     

Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system

We have found two novel lipocalins in the fruit fly Drosophila melanogaster that are homologous to the grasshopper Lazarillo, a singular lipocalin within this protein family which functions in axon guidance during nervous system development. Sequence analysis suggests that the two Drosophila proteins are secreted and possess peptide regions unique in the lipocalin family. The mRNAs of DNLaz (for Drosophila neural Lazarillo) and DGLaz (for Drosophila glial Lazarillo) are expressed with different temporal patterns during embryogenesis. They show low levels of larval expression and are highly expressed in pupa and adult flies. DNLaz mRNA is transcribed in a subset of neurons and neuronal precursors in the embryonic CNS. DGLaz mRNA is found in a subset of glial cells of the CNS: the longitudinal glia and the medial cell body glia. Both lipocalins are also expressed outside the nervous system in the developing gut, fat body and amnioserosa. The DNLaz protein is detected in a subset of axons in the developing CNS. Treatment with a secretion blocker enhances the antibody labeling, indicating the DNLaz secreted nature. These findings make the embryonic nervous system expression of lipocalins a feature more widespread than previously thought. We propose that DNLaz and DGLaz may have a role in axonal outgrowth and pathfinding, although other putative functions are also discussed.     

Draper-mediated and phosphatidylserine-independent phagocytosis of apoptotic cells by Drosophila hemocytes/macrophages

The mechanism of phagocytic elimination of dying cells in Drosophila is poorly understood. This study was undertaken to examine the recognition and engulfment of apoptotic cells by Drosophila hemocytes/macrophages in vitro and in vivo. In the in vitro analysis, l(2)mbn cells (a cell line established from larval hemocytes of a tumorous Drosophila mutant) were used as phagocytes. When l(2)mbn cells were treated with the molting hormone 20-hydroxyecdysone, the cells acquired the ability to phagocytose apoptotic S2 cells, another Drosophila cell line. S2 cells undergoing cycloheximide-induced apoptosis exposed phosphatidylserine on their surface, but their engulfment by l(2)mbn cells did not seem to be mediated by phosphatidylserine. The level of Croquemort, a candidate phagocytosis receptor of Drosophila hemocytes/macrophages, increased in l(2)mbn cells after treatment with 20-hydroxyecdysone, whereas that of Draper, another candidate phagocytosis receptor, remained unchanged. However, apoptotic cell phagocytosis was reduced when the expression of Draper, but not of Croquemort, was inhibited by RNA interference in hormone-treated l(2)mbn cells. We next examined whether Draper is responsible for the phagocytosis of apoptotic cells in vivo using an assay for engulfment based on assessing DNA degradation of apoptotic cells in dICAD mutant embryos (which only occurred after ingestion by the phagocytes). RNA interference-mediated decrease in the level of Draper in embryos of mutant flies was accompanied by a decrease in the number of cells containing fragmented DNA. Furthermore, histochemical analyses of dispersed embryonic cells revealed that the level of phagocytosis of apoptotic cells by hemocytes/macrophages was reduced when Draper expression was inhibited. These results indicate that Drosophila hemocytes/macrophages execute Draper-mediated phagocytosis to eliminate apoptotic cells.     

Lazarillo, a neuronal lipocalin in grasshoppers with a role in axon guidance

In this report we present a review on the grasshopper lipocalin Lazarillo with special emphasis on how its molecular properties could account for its known function: the guidance of pioneer neurons during nervous system development. The expression and function of Lazarillo in a subset of developing neurons, its heavy glycosylation and its glycosylphosphatidylinositol linkage to the plasma membrane, make Lazarillo a unique member of the lipocalin family. We have built a model of the tertiary structure of Lazarillo in which we have studied the exposed surfaces in search for clues about ligand and protein interactions with Lazarillo. Our hypotheses about how this lipocalin can exert its function are discussed.     

Specific cell ablation in Drosophila using the toxic viral protein M2(H37A)

The expression of toxic viral proteins for the purpose of eliminating distinct populations of cells, while leaving the rest of an organism unaffected, is a valuable method for analyzing development. Using the Gal4-UAS system, we employed the M2(H37A) toxic ion channel of the influenza-A virus to selectively ablate the Drosophila eye-antennal imaginal discs, hemocytes, dorsal vessel and nervous tissue, and comparatively monitored the effects of expressing the apoptosis-promoting protein Reaper in identical cell populations. In this report, we demonstrate the effectiveness of M2(H37A)-mediated ablation as a new means to selectively eliminate cells of interest during Drosophila development.     

Sequence analysis and neuronal expression of fasciclin I in grasshopper and Drosophila

The fasciclin I, II, and III glycoproteins are expressed on different subsets of axon bundles (fascicles) in insect embryos and are thus candidates for surface recognition molecules involved in growth cone guidance. Here we present the sequence of grasshopper fasciclin I and the identification and sequence of the Drosophila fasciclin I homolog. In both species, fasciclin I appears to be an extrinsic membrane protein with a signal sequence but no transmembrane region; the protein comprises four homologous domains of approximately 150 amino acids each. Antibodies against Drosophila fasciclin I reveal that it is expressed on the surface of a subset of commissural axon pathways in the embryonic central nervous system and on all sensory axon pathways in the peripheral nervous system. This pattern of expression is similar to that in grasshopper.     

Slob, a Slowpoke channel-binding protein, modulates synaptic transmission

Modulation of ion channels by regulatory proteins within the same macromolecular complex is a well-accepted concept, but the physiological consequences of such modulation are not fully understood. Slowpoke (Slo), a potassium channel critical for action potential repolarization and transmitter release, is regulated by Slo channel-binding protein (Slob), a Drosophila melanogaster Slo (dSlo) binding partner. Slob modulates the voltage dependence of dSlo channel activation in vitro and exerts similar effects on the dSlo channel in Drosophila central nervous system neurons in vivo. In addition, Slob modulates action potential duration in these neurons. Here, we investigate further the functional consequences of the modulation of the dSlo channel by Slob in vivo, by examining larval neuromuscular synaptic transmission in flies in which Slob levels have been altered. In Slob-null flies generated through P-element mutagenesis, as well as in Slob knockdown flies generated by RNA interference (RNAi), we find an enhancement of synaptic transmission but no change in the properties of the postsynaptic muscle cell. Using targeted transgenic rescue and targeted expression of Slob-RNAi, we find that Slob expression in neurons (but not in the postsynaptic muscle cell) is critical for its effects on synaptic transmission. Furthermore, inhibition of dSlo channel activity abolishes these effects of Slob. These results suggest that presynaptic Slob, by regulating dSlo channel function, participates in the modulation of synaptic transmission.     

Pax group III genes and the evolution of insect pair-rule patterning

Pair-rule genes were identified and named for their role in segmentation in embryos of the long germ insect Drosophila. Among short germ insects these genes exhibit variable expression patterns during segmentation and thus are likely to play divergent roles in this process. Understanding the details of this variation should shed light on the evolution of the genetic hierarchy responsible for segmentation in Drosophila and other insects. We have investigated the expression of homologs of the Drosophila Pax group III genes paired, gooseberry and gooseberry-neuro in short germ flour beetles and grasshoppers. During Drosophila embryogenesis, paired acts as one of several pair-rule genes that define the boundaries of future parasegments and segments, via the regulation of segment polarity genes such as gooseberry, which in turn regulates gooseberry-neuro, a gene expressed later in the developing nervous system. Using a crossreactive antibody, we show that the embryonic expression of Pax group III genes in both the flour beetle Tribolium and the grasshopper Schistocerca is remarkably similar to the pattern in Drosophila. We also show that two Pax group III genes, pairberry1 and pairberry2, are responsible for the observed protein pattern in grasshopper embryos. Both pairberry1 and pairberry2 are expressed in coincident stripes of a one-segment periodicity, in a manner reminiscent of Drosophila gooseberry and gooseberry-neuro. pairberry1, however, is also expressed in stripes of a two-segment periodicity before maturing into its segmental pattern. This early expression of pairberry1 is reminiscent of Drosophila paired and represents the first evidence for pair-rule patterning in short germ grasshoppers or any hemimetabolous insect.     

glide/gcmIs Expressed and Required in the Scavenger Cell Lineage

Glial cell differentiation inDrosophila melanogasterrequires the activity ofglide/gcm(glial cell deficient/glial cell missing). The role of this gene is to direct the cell fate switch between neurons and glial cells by activating the glial developmental program in multipotent precursor cells of the nervous system. In this paper, we show thatglide/gcmis also expressed and required in the lineage of hemocytes/macrophages, scavenger cells that phagocytose cells undergoing programmed cell death. In addition, we show that, as for glial cells,glide/gcmplays an instructive role in hemocyte differentiation. Interestingly, it has been shown that in the development of the fly adult nervous system the role of scavenger cells is played by glial cells. These data and our findings on the dual role ofglide/gcmindicate that glial cells and hemocytes/macrophages are functionally and molecularly related.     

Of blood cells and the nervous system

Hematopoiesis is well-conserved between Drosophila and vertebrates. Similar as in vertebrates, the sites of hematopoiesis shift during Drosophila development. Blood cells (hemocytes) originate de novo during hematopoietic waves in the embryo and in the Drosophila lymph gland. In contrast, the hematopoietic wave in the larva is based on the colonization of resident hematopoietic sites by differentiated hemocytes that arise in the embryo, much like in vertebrates the colonization of peripheral tissues by primitive macrophages of the yolk sac, or the seeding of fetal liver, spleen and bone marrow by hematopoietic stem and progenitor cells. At the transition to the larval stage, Drosophila embryonic hemocytes retreat to hematopoietic “niches,” i.e., segmentally repeated hematopoietic pockets of the larval body wall that are jointly shared with sensory neurons and other cells of the peripheral nervous system (PNS). Hemocytes rely on the PNS for their localization and survival, and are induced to proliferate in these microenvironments, expanding to form the larval hematopoietic system. In this process, differentiated hemocytes from the embryo resume proliferation and self-renew, omitting the need for an undifferentiated prohemocyte progenitor. Larval hematopoiesis is the first Drosophila model for blood cell colonization and niche support by the PNS. It suggests an interface where innocuous or noxious sensory inputs regulate blood cell homeostasis or immune responses. The system adds to the growing concept of nervous system dependence of hematopoietic microenvironments and organ stem cell niches, which is being uncovered across phyla.     

Condensation of the central nervous system in embryonic Drosophila is inhibited by blocking hemocyte migration or neural activity

Condensation is a process whereby a tissue undergoes a coordinated decrease in size and increase in cellular density during development. Although it occurs in many developmental contexts, the mechanisms underlying this process are largely unknown. Here, we investigate condensation in the embryonic Drosophila ventral nerve cord (VNC). Two major events coincide with condensation during embryogenesis: the deposition of extracellular matrix by hemocytes, and the onset of central nervous system activity. We find that preventing hemocyte migration by removing the function of the Drosophila VEGF receptor homologue, Pvr, or by disrupting Rac1 function in these cells, inhibits condensation. In the absence of hemocytes migrating adjacent to the developing VNC, the extracellular matrix components Collagen IV, Viking and Peroxidasin are not deposited around this tissue. Blocking neural activity by targeted expression of tetanus toxin light chain or an inwardly rectifying potassium channel also inhibits condensation. We find that disrupting Rac1 function in either glia or neurons, including those located in the nerve cord, causes a similar phenotype. Our data suggest that condensation of the VNC during Drosophila embryogenesis depends on both hemocyte-deposited extracellular matrix and neural activity, and allow us to propose a mechanism whereby these processes work together to shape the developing central nervous system.     

Localization-dependent and -independent roles of numb contribute to cell-fate specification in Drosophila

During asymmetric cell division, protein determinants are segregated into one of the two daughter cells. The Numb protein acts as a segregating determinant during both mouse and Drosophila development. In flies, Numb localizes asymmetrically and is required for cell-fate specification in the central and peripheral nervous systems, as well as during muscle and heart development. Whether its asymmetric segregation is important to the performance of these functions is not firmly established. Here, we demonstrate that Numb acts both in a localization-dependent and in a localization-independent manner. We have generated numb mutants that affect only the asymmetric localization of the protein during mitosis. We demonstrate that asymmetric segregation of Numb into one of the two daughter cells is absolutely essential for cell-fate specification in the Drosophila peripheral nervous system. Numb localization is also essential in MP2 neuroblasts in the central nervous system and during muscle development. Surprisingly, in dividing ganglion mother cells or during heart development, Numb function is independent of its ability to segregate asymmetrically in mitosis. Our results suggest that two classes of asymmetric cell division exist, each with different requirements for asymmetric inheritance of cell-fate determinants.     

Expression of engrailed proteins in arthropods, annelids, and chordates

engrailed is a homeobox gene that has an important role in Drosophila segmentation. Genes homologous to engrailed have been identified in several other organisms. Here we describe a monoclonal antibody that recognizes a conserved epitope in the homeodomain of engrailed proteins of a number of different arthropods, annelids, and chordates; we use this antibody to isolate the grasshopper engrailed gene. In Drosophila embryos, the antibody reveals engrailed protein in the posterior portion of each segment during segmentation, and in a segmentally reiterated subset of neuronal cells during neurogenesis. Other arthropods, including grasshopper and two crustaceans, have similar patterns of engrailed expression. However, these patterns of expression are not shared by the annelids or chordates we examined. Our results provide the most comprehensive view that has been obtained of how expression patterns of a regulatory gene vary during evolution. On the basis of these patterns, we suggest that engrailed is a gene whose ancestral function was in neurogenesis and whose function was co-opted during the evolution of segmentation in the arthropods, but not in the annelids and chordates.     

Single-cell RNA sequencing identifies novel cell types in Drosophila blood

Drosophila has been extensively used to model the human blood-immune system,as both systems share many developmental and immune response mechanisms.However,while many human blood cell types have been identified,only three were found in flies:plasmatocytes,crystal cells and lamellocytes.To better understand the complexity of fly blood system,we used single-cell RNA sequencing technology to generate co mprehensive gene expression profiles for Drosophila circulating blood cells.In addition to the known cell types,we identified two new Drosophila blood cell types:thanacytes and primocytes.Thanacytes,which express many stimulus response genes,are involved in distinct responses to different types of bacteria.Primocytes,which express cell fate commitment and signaling genes,appear to be involved in keeping stem cells in the circulating blood.Furthermore,our data revealed four novel plasmatocyte subtypes(Ppn+,CAH7~+,Lsp~+ and reservoir plasmatocytes),each with unique molecular identities and distinct predicted functions.We also identified cross-species markers from Drosophila hemocytes to human blood cells.Our analysis unveiled a more complex Drosophila blood system and broadened the scope of using Drosophila to model human blood system in development and disease.     

Developmental expression and molecular characterization of two gap junction channel proteins expressed during embryogenesis in the grasshopper Schistocerca americana

Gap junctions are membrane channels that directly connect the cytoplasm of neighboring cells, allowing the exchange of ions and small molecules. Two analogous families of proteins, the connexins and innexins, are the channel-forming molecules in vertebrates and invertebrates, respectively. In order to study the role of gap junctions in the embryonic development of the nervous system, we searched for innexins in the grasshopper Schistocerca americana. Here we present the molecular cloning and sequence analysis of two novel innexins, G-Inx(1) and G-Inx(2), expressed during grasshopper embryonic development. The analysis of G-Inx(1) and G-Inx(2) proteins suggests they bear four transmembrane domains, which show strong conservation in members of the innexin family. The study of the phylogenetic relationships between members of the innexin family and the new grasshopper proteins suggests that G-Inx(1) is orthologous to the Drosophila 1(1)-ogre. However, G-Inx(2) seems to be a member of a new group of insect innexins. We used in situ hybridization with the G-Inx(1) and G-Inx(2) cDNA clones, and two polyclonal sera raised against different regions of G-Inx(1) to study the mRNA and protein expression patterns and the subcellular localization of the grasshopper innexins. G-Inx(1) is primarily expressed in the embryonic nervous system, in neural precursors and glial cells. In addition, a restricted stripe of epithelial cells in the developing limb, involved in the guidance of sensory growth cones, expresses G-Inx(1). G-Inx(2) expression is more widespread in the grasshopper embryo, but a restricted expression is found in a subset of neural precursors. The generally different but partially overlapping expression patterns of G-Inx(1) and G-Inx(2) supports the combinatorial character of gap junction formation in invertebrates, an essential property to generate specificity in this form of cell-cell communication.     

Nanos plays a conserved role in axial patterning outside of the Diptera

Axial patterning is a fundamental event in early development, and molecules involved in determining the body axes provide a coordinate system for subsequent patterning. While orthologs of Drosophila bicoid and nanos play a conserved role in anteroposterior (AP) patterning within at least a subset of Diptera, conservation of this process has not yet been demonstrated outside of the flies. Indeed, it has been argued that bicoid, an instrumental "anterior" factor in Drosophila melanogaster, acquired this role during the evolution of more-derived dipterans. Interestingly, the interaction of Drosophila maternal nanos and maternal hunchback provides a system for patterning the AP axis that is partially redundant to the anterior system. Previous studies in grasshoppers suggest that hunchback may play a conserved role in axial patterning in this insect, but this function may be supplied solely by the zygotic component of hunchback expression. Here we provide evidence that the early pattern of zygotic grasshopper Hunchback expression is achieved through translational repression that may be mediated through the action of grasshopper nanos. This is consistent with the notion that an anterior gradient system is not necessary in all insects and that the posterior pole "probably conveys longitudinal polarity on the ensuing germ anlage".     

Specific cell ablation in Drosophila using the toxic viral protein M2(H37A)

The expression of toxic viral proteins for the purpose of eliminating distinct populations of cells, while leaving the rest of an organism unaffected, is a valuable method for analyzing development. Using the Gal4-UAS system, we employed the M2(H37A) toxic ion channel of the influenza-A virus to selectively ablate the Drosophila eye-antennal imaginal discs, hemocytes, dorsal vessel and nervous tissue and comparatively monitored the effects of expressing the apoptosis-promoting protein Reaper in identical cell populations. In this report, we demonstrate the effectiveness of M2(H37A)-mediated ablation as a new means to selectively eliminate cells of interest during Drosophila development.Key words: cell ablation, dorsal vessel, Drosophila, eye imaginal disc, hand, lamellocytes, M2 toxin, reaper     

Characterization of a novel Drosophila melanogaster galectin. Expression in developing immune, neural, and muscle tissues.

We have cloned and characterized the first galectin to be identified in Drosophila melanogaster. The amino acid sequence of Drosophila galectin showed striking sequence similarity to invertebrate and vertebrate galectins and contained amino acids that are crucial for binding beta-galactoside sugars. Confirming its identity as a galectin family member, the Drosophila galectin bound beta-galactoside sugars. Structurally, the Drosophila galectin was a tandem repeat galectin containing two carbohydrate recognition domains connected by a unique peptide link. This divalent structure suggests that like mammalian galectins, Drosophila galectin may mediate cell-cell communication or facilitate cross-linking of receptors to trigger signal transduction events. The Drosophila galectin was very abundant in embryonic, larval, and adult Drosophila. During embryogenesis, Drosophila galectin had a unique and specific tissue distribution. Drosophila galectin expression was concentrated in somatic and visceral musculature and in the central nervous system. Similar to other insect lectins, Drosophila galectin may function in both embryogenesis and in host defense. Drosophila galectin was expressed by hemocytes, circulating phagocytic cells, suggesting a role for Drosophila galectin in the innate immune system.