Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila post-embryonic brain

Glial cells are essential for the development and function of the nervous system. In the mammalian brain, vast numbers of glia of several different functional types are generated during late embryonic and early foetal development. However, the molecular cues that instruct gliogenesis and determine glial cell type are poorly understood. During post-embryonic development, the number of glia in the Drosophila larval brain increases dramatically, potentially providing a powerful model for understanding gliogenesis. Using glial-specific clonal analysis we find that perineural glia and cortex glia proliferate extensively through symmetric cell division in the post-embryonic brain. Using pan-glial inhibition and loss-of-function clonal analysis we find that Insulin-like receptor (InR)/Target of rapamycin (TOR) signalling is required for the proliferation of perineural glia. Fibroblast growth factor (FGF) signalling is also required for perineural glia proliferation and acts synergistically with the InR/TOR pathway. Cortex glia require InR in part, but not downstream components of the TOR pathway, for proliferation. Moreover, cortex glia absolutely require FGF signalling, such that inhibition of the FGF pathway almost completely blocks the generation of cortex glia. Neuronal expression of the FGF receptor ligand Pyramus is also required for the generation of cortex glia, suggesting a mechanism whereby neuronal FGF expression coordinates neurogenesis and cortex gliogenesis. In summary, we have identified two major pathways that control perineural and cortex gliogenesis in the post-embryonic brain and have shown that the molecular circuitry required is lineage specific

Relative Roles of the Cellular and Humoral Responses in the Drosophila Host Defense against Three Gram-Positive Bacterial Infections

BACKGROUND: Two NF-kappaB signaling pathways, Toll and immune deficiency (imd), are required for survival to bacterial infections in Drosophila. In response to septic injury, these pathways mediate rapid transcriptional activation of distinct sets of effector molecules, including antimicrobial peptides, which are important components of a humoral defense response. However, it is less clear to what extent macrophage-like hemocytes contribute to host defense. METHODOLOGY/PRINCIPAL FINDINGS: In order to dissect the relative importance of humoral and cellular defenses after septic injury with three different gram-positive bacteria (Micrococcus luteus, Enterococcus faecalis, Staphylococcus aureus), we used latex bead pre-injection to ablate macrophage function in flies wildtype or mutant for various Toll and imd pathway components. We found that in all three infection models a compromised phagocytic system impaired fly survival--independently of concomitant Toll or imd pathway activation. Our data failed to confirm a role of the PGRP-SA and GNBP1 Pattern Recognition Receptors for phagocytosis of S. aureus. The Drosophila scavenger receptor Eater mediates the phagocytosis by hemocytes or S2 cells of E. faecalis and S. aureus, but not of M. luteus. In the case of M. luteus and E. faecalis, but not S. aureus, decreased survival due to defective phagocytosis could be compensated for by genetically enhancing the humoral immune response. CONCLUSIONS/SIGNIFICANCE: Our results underscore the fundamental importance of both cellular and humoral mechanisms in Drosophila immunity and shed light on the balance between these two arms of host defense depending on the invading pathogen

Effective but Costly, Evolved Mechanisms of Defense against a Virulent Opportunistic Pathogen in Drosophila melanogaster

Drosophila harbor substantial genetic variation for antibacterial defense, and investment in immunity is thought to involve a costly trade-off with life history traits, including development, life span, and reproduction. To understand the way in which insects invest in fighting bacterial infection, we selected for survival following systemic infection with the opportunistic pathogen Pseudomonas aeruginosa in wild-caught Drosophila melanogaster over 10 generations. We then examined genome-wide changes in expression in the selected flies relative to unselected controls, both of which had been infected with the pathogen. This powerful combination of techniques allowed us to specifically identify the genetic basis of the evolved immune response. In response to selection, population-level survivorship to infection increased from 15% to 70%. The evolved capacity for defense was costly, however, as evidenced by reduced longevity and larval viability and a rapid loss of the trait once selection pressure was removed. Counter to expectation, we observed more rapid developmental rates in the selected flies. Selection-associated changes in expression of genes with dual involvement in developmental and immune pathways suggest pleiotropy as a possible mechanism for the positive correlation. We also found that both the Toll and the Imd pathways work synergistically to limit infectivity and that cellular immunity plays a more critical role in overcoming P. aeruginosa infection than previously reported. This work reveals novel pathways by which Drosophila can survive infection with a virulent pathogen that may be rare in wild populations, however, due to their cost

Bacterial Communities of Diverse Drosophila Species: Ecological Context of a Host–Microbe Model System

Drosophila melanogaster is emerging as an important model of non-pathogenic host–microbe interactions. The genetic and experimental tractability of Drosophila has led to significant gains in our understanding of animal–microbial symbiosis. However, the full implications of these results cannot be appreciated without the knowledge of the microbial communities associated with natural Drosophila populations. In particular, it is not clear whether laboratory cultures can serve as an accurate model of host–microbe interactions that occur in the wild, or those that have occurred over evolutionary time. To fill this gap, we characterized natural bacterial communities associated with 14 species of Drosophila and related genera collected from distant geographic locations. To represent the ecological diversity of Drosophilids, examined species included fruit-, flower-, mushroom-, and cactus-feeders. In parallel, wild host populations were compared to laboratory strains, and controlled experiments were performed to assess the importance of host species and diet in shaping bacterial microbiome composition. We find that Drosophilid flies have taxonomically restricted bacterial communities, with 85% of the natural bacterial microbiome composed of only four bacterial families. The dominant bacterial taxa are widespread and found in many different host species despite the taxonomic, ecological, and geographic diversity of their hosts. Both natural surveys and laboratory experiments indicate that host diet plays a major role in shaping the Drosophila bacterial microbiome. Despite this, the internal bacterial microbiome represents only a highly reduced subset of the external bacterial communities, suggesting that the host exercises some level of control over the bacteria that inhabit its digestive tract. Finally, we show that laboratory strains provide only a limited model of natural host–microbe interactions. Bacterial taxa used in experimental studies are rare or absent in wild Drosophila populations, while the most abundant associates of natural Drosophila populations are rare in the lab

The Drosophila melanogaster host model

The deleterious and sometimes fatal outcomes of bacterial infectious diseases are the net result of the interactions between the pathogen and the host, and the genetically tractable fruit fly, Drosophila melanogaster, has emerged as a valuable tool for modeling the pathogen–host interactions of a wide variety of bacteria. These studies have revealed that there is a remarkable conservation of bacterial pathogenesis and host defence mechanisms between higher host organisms and Drosophila. This review presents an in-depth discussion of the Drosophila immune response, the Drosophila killing model, and the use of the model to examine bacterial–host interactions. The recent introduction of the Drosophila model into the oral microbiology field is discussed, specifically the use of the model to examine Porphyromonas gingivalis–host interactions, and finally the potential uses of this powerful model system to further elucidate oral bacterial-host interactions are addressed

Methods to Quantify In Vivo Phagocytic Uptake and Opsonization of Live or Killed Microbes in Drosophila melanogaster

International audienc

Anthrax lethal factor and edema factor act on conserved targets in Drosophila

Many bacterial toxins act on conserved components of essential host-signaling pathways. One consequence of this conservation is that genetic model organisms such as Drosophila melanogaster can be used for analyzing the mechanism of toxin action. In this study, we characterize the activities of two anthrax virulence factors, lethal factor (LF) and edema factor, in transgenic Drosophila. LF is a zinc metalloprotease that cleaves and inactivates most human mitogen-activated protein kinase (MAPK) kinases (MAPKKs). We found that LF similarly cleaves the Drosophila MAPK kinases Hemipterous (Hep) and Licorne in vitro. Consistent with these observations, expression of LF in Drosophila inhibited the Hep/c-Jun N-terminal kinase pathway during embryonic dorsal closure and the related process of adult thoracic closure. Epistasis experiments confirmed that LF acts at the level of Hep. We also found that LF inhibits Ras/MAPK signaling during wing development and that LF acts upstream of MAPK and downstream of Raf, consistent with LF acting at the level of Dsor. In addition, we found that edema factor, a potent adenylate cyclase, inhibits the hh pathway during wing development, consistent with the known role of cAMP-dependent PKA in suppressing the Hedgehog response. These results demonstrate that anthrax toxins function in Drosophila as they do in mammalian cells and open the way to using Drosophila as a multicellular host system for studying the in vivo function of diverse toxins and virulence factors

Recognition of distinct RNA motifs by the clustered CCCH zinc fingers of neuronal protein Unkempt

Unkempt is an evolutionarily conserved RNA-binding protein that regulates translation of its target genes and is required for the establishment of the early bipolar neuronal morphology. Here we determined the X-ray crystal structure of mouse Unkempt and show that its six CCCH zinc fingers (ZnFs) form two compact clusters, ZnF–3 and ZnF4–6, that recognize distinct trinucleotide RNA substrates. Both ZnF clusters adopt a similar overall topology and use distinct recognition principles to target specific RNA sequences. Structure-guided point mutations reduce the RNA binding affinity of Unkempt both in vitro and in vivo, ablate Unkempt’s translational control and impair the ability of Unkempt to induce a bipolar cellular morphology. Our study unravels a new mode of RNA sequence recognition by clusters of CCCH ZnFs that is critical for post-transcriptional control of neuronal morphology