Two Independent Functions of Collier/Early B Cell Factor in the Control of Drosophila Blood Cell Homeostasis

Blood cell production in the Drosophila hematopoietic organ, the lymph gland, is controlled by intrinsic factors and extrinsic signals. Initial analysis of Collier/Early B Cell Factor function in the lymph gland revealed the role of the Posterior Signaling Center (PSC) in mounting a dedicated cellular immune response to wasp parasitism. Further, premature blood cell differentiation when PSC specification or signaling was impaired, led to assigning the PSC a role equivalent to the vertebrate hematopoietic niche. We report here that Collier is expressed in a core population of lymph gland progenitors and cell autonomously maintains this population. The PSC contributes to lymph gland homeostasis by regulating blood cell differentiation, rather than by maintaining core progenitors. In addition to PSC signaling, switching off Collier expression in progenitors is required for efficient immune response to parasitism. Our data show that two independent sites of Collier/Early B Cell Factor expression, hematopoietic progenitors and the PSC, achieve control of hematopoiesis.     

Dual role for Insulin/TOR signaling in the control of hematopoietic progenitor maintenance in Drosophila

The interconnected Insulin/IGF signaling (IlS) and Target of Rapamycin (TOR) signaling pathways constitute the main branches of the nutrient-sensing system that couples growth to nutritional conditions in Drosophila. Here, we addressed the influence of these pathways and of diet restriction on the balance between the maintenance of multipotent hematopoietic progenitors and their differentiation in the Drosophila lymph gland. In this larval hematopoietic organ, a pool of stem-like progenitor blood cells (prohemocytes) is kept undifferentiated in response to signaling from a specialized group of cells forming the posterior signaling center (PSC), which serves as a stem cell niche. We show that, reminiscent of the situation in human, loss of the negative regulator of IIS Pten results in lymph gland hyperplasia, aberrant blood cell differentiation and hematopoietic progenitor exhaustion. Using site-directed loss- and gain-of-function analysis, we demonstrate that components of the IIS/TOR pathways control lymph gland homeostasis at two levels. First, they cell-autonomously regulate the size and activity of the hematopoietic niche. Second, they are required within the prohemocytes to control their growth and maintenance. Moreover, we show that diet restriction or genetic alteration mimicking amino acid deprivation triggers progenitor cell differentiation. Hence, our study highlights the role of the IIS/TOR pathways in orchestrating hematopoietic progenitor fate and links blood cell fate to nutritional status.     

Neuron-Specific Feeding RNAi in C. elegans and Its Use in a Screen for Essential Genes Required for GABA Neuron Function

Forward genetic screens are important tools for exploring the genetic requirements for neuronal function. However, conventional forward screens often have difficulty identifying genes whose relevant functions are masked by pleiotropy. In particular, if loss of gene function results in sterility, lethality, or other severe pleiotropy, neuronal-specific functions cannot be readily analyzed. Here we describe a method in C. elegans for generating cell-specific knockdown in neurons using feeding RNAi and its application in a screen for the role of essential genes in GABAergic neurons. We combine manipulations that increase the sensitivity of select neurons to RNAi with manipulations that block RNAi in other cells. We produce animal strains in which feeding RNAi results in restricted gene knockdown in either GABA-, acetylcholine-, dopamine-, or glutamate-releasing neurons. In these strains, we observe neuron cell-type specific behavioral changes when we knock down genes required for these neurons to function, including genes encoding the basal neurotransmission machinery. These reagents enable high-throughput, cell-specific knockdown in the nervous system, facilitating rapid dissection of the site of gene action and screening for neuronal functions of essential genes. Using the GABA-specific RNAi strain, we screened 1,320 RNAi clones targeting essential genes on chromosomes I, II, and III for their effect on GABA neuron function. We identified 48 genes whose GABA cell-specific knockdown resulted in reduced GABA motor output. This screen extends our understanding of the genetic requirements for continued neuronal function in a mature organism.     

A parasitoid wasp of Drosophila employs preemptive and reactive strategies to deplete its host’s blood cells

The wasps Leptopilina heterotoma parasitize and ingest their Drosophila hosts. They produce extracellular vesicles (EVs) in the venom that are packed with proteins, some of which perform immune suppressive functions. EV interactions with blood cells of host larvae are linked to hematopoietic depletion, immune suppression, and parasite success. But how EVs disperse within the host, enter and kill hematopoietic cells is not well understood. Using an antibody marker for L. heterotoma EVs, we show that these parasite-derived structures are readily distributed within the hosts’ hemolymphatic system. EVs converge around the tightly clustered cells of the posterior signaling center (PSC) of the larval lymph gland, a small hematopoietic organ in Drosophila. The PSC serves as a source of developmental signals in naïve animals. In wasp-infected animals, the PSC directs the differentiation of lymph gland progenitors into lamellocytes. These lamellocytes are needed to encapsulate the wasp egg and block parasite development. We found that L. heterotoma infection disassembles the PSC and PSC cells disperse into the disintegrating lymph gland lobes. Genetically manipulated PSC-less lymph glands remain non-responsive and largely intact in the face of L. heterotoma infection. We also show that the larval lymph gland progenitors use the endocytic machinery to internalize EVs. Once inside, L. heterotoma EVs damage the Rab7- and LAMP-positive late endocytic and phagolysosomal compartments. Rab5 maintains hematopoietic and immune quiescence as Rab5 knockdown results in hematopoietic over-proliferation and ectopic lamellocyte differentiation. Thus, both aspects of anti-parasite immunity, i.e., (a) phagocytosis of the wasp’s immune-suppressive EVs, and (b) progenitor differentiation for wasp egg encapsulation reside in the lymph gland. These results help explain why the lymph gland is specifically and precisely targeted for destruction. The parasite’s simultaneous and multipronged approach to block cellular immunity not only eliminates blood cells, but also tactically blocks the genetic programming needed for supplementary hematopoietic differentiation necessary for host success. In addition to its known functions in hematopoiesis, our results highlight a previously unrecognized phagocytic role of the lymph gland in cellular immunity. EV-mediated virulence strategies described for L. heterotoma are likely to be shared by other parasitoid wasps; their understanding can improve the design and development of novel therapeutics and biopesticides as well as help protect biodiversity.     

Pupal RNA interference methods for analyzing adult development in stag beetles

Holometabolous insects dramatically change their morphology via molt, both from larva to pupa and from pupa to adult. In nonmodel insects, RNA interference (RNAi) is a strong tool for analyzing gene function during postembryonic development. In many cases, larval RNAi is effective for analyzing genes involved in morphogenesis via metamorphosis. However, RNAi of genes involved in development sometimes results in lethality before animals metamorphose to pupae and/or adults, making it impossible to analyze their function during the pupal period. In this study, we establish a pupal RNAi system in the stag beetle Dorcus rectus. We selected the genes white and scarlet for RNAi knockdown to investigate appropriate injection timing and position. Both genes are known to be involved in eye pigmentation. By using these candidate genes, we demonstrate the potential of pupal RNAi in this experimental system. This method will be useful for analyzing pupal-specific morphogenesis including fine-shaping of the enlarged male mandible in this species.     

Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.     

Sensitivity to RNA interference-mediated gene silencing in intact and ligated larvae of the armyworm, Mythimna separata (Lepidoptera: Noctuidae)

RNA interference (RNAi) is a common tool for analysis of gene function in both model and non-model insects, but it is becoming evident that RNAi efficiency varies considerably from species to species. We examined RNAi efficiency in larvae of the armyworm Mythimna separata (Walker) using multiple genes and tissues. First, we showed that five different target genes exhibited distinct tissue distribution patterns by quantitative determination of mRNA in total hemocytes, foregut, midgut, hindgut, Malpighian tubules and fat body: neuroglian mRNA was most abundant in fat body; inhibitor of apoptosis proteins mRNA was found to be ubiquitous; aquaporin 4 mRNA was most enriched in hindgut; cueball and prophenoloxidase 2 were mainly expressed in hemocytes. Second, we assessed sensitivity to gene silencing by double-strand RNA injection of these five genes in the six different tissues. We found that these genes generally showed refractoriness to double-strand RNA-mediated gene knockdown irrespective of the tissue tested. Finally, we demonstrated that appreciable gene knockdown was achieved at least in the adhering hemocyte fraction when larval isolated abdomen was prepared by ligation and subjected to dsRNA injection. Our study thus added detailed information on the refractoriness of larval tissues of a lepidopteran insect to gene silencing through RNAi and provided a new potential approach to improve RNAi efficiency.     

Dissecting the Biological Role of Mucin-type O-Glycosylation Using RNA Interference in Drosophila Cell Culture

Mucin type O-glycosylation is a highly conserved form of post-translational modification initiated by the family of enzymes known as the polypeptide α-N-acetylgalactosaminyltransferases (ppGalNAcTs in mammals and PGANTs in Drosophila). To address the cellular functions of the many PGANT family members, RNA interference (RNAi) to each pgant gene was performed in two independent Drosophila cell culture lines. We demonstrate that RNAi to individual pgant genes results in specific reduction in gene expression without affecting the expression of other family members. Cells with reduced expression of individual pgant genes were then examined for changes in viability, morphology, adhesion, and secretion to assess the contribution of each family member to these cellular functions. Here we find that RNAi to pgant3, pgant6, or pgant7 resulted in reduced secretion, further supporting a role for O-glycosylation in proper secretion. Additionally, RNAi to pgant3 or pgant6 resulted in altered Golgi organization, suggesting a role for each in establishing or maintaining proper secretory apparatus structure. Other subcellular effects observed included multinucleated cells seen after RNAi to either pgant2 or pgant35A, suggesting a role for these genes in the completion of cytokinesis. These studies demonstrate the efficient and specific knockdown of pgant gene expression in two Drosophila cell culture systems, resulting in specific morphological and functional effects. Our work provides new information regarding the biological roles of O-glycosylation and illustrates a new platform for interrogating the cellular and subcellular effects of this form of post-translational modification.     

Polydnaviral Ankyrin Proteins Aid Parasitic Wasp Survival by Coordinate and Selective Inhibition of Hematopoietic and Immune NF-kappa B Signaling in Insect Hosts

Polydnaviruses are mutualists of their parasitoid wasps and express genes in immune cells of their Lepidopteran hosts. Polydnaviral genomes carry multiple copies of viral ankyrins or vankyrins. Vankyrin proteins are homologous to IκB proteins, but lack sequences for regulated degradation. We tested if Ichnoviral Vankyrins differentially impede Toll-NF-κB-dependent hematopoietic and immune signaling in a heterologous in vivo Drosophila, system. We first show that hematopoiesis and the cellular encapsulation response against parasitoid wasps are tightly-linked via NF-κB signaling. The niche, which neighbors the larval hematopoietic progenitors, responds to parasite infection. Drosophila NF-κB proteins are expressed in the niche, and non cell-autonomously influence fate choice in basal and parasite-activated hematopoiesis. These effects are blocked by the Vankyrin I²-vank-3, but not by P-vank-1, as is the expression of a NF-κB target transgene. I²-vank-3 and P-vank-1 differentially obstruct cellular and humoral inflammation. Additionally, their maternal expression weakens ventral embryonic patterning. We propose that selective perturbation of NF-κB-IκB interactions in natural hosts of parasitic wasps negatively impacts the outcome of hematopoietic and immune signaling and this immune deficit contributes to parasite survival and species success in nature.     

An Altered Method of Feeding RNAi That Knocks Down Multiple Genes Simultaneously in the Nematode Caenorhabditis elegans

In reverse genetics, RNA interference (RNAi) which is substitutable for gene-disruption, is an outstanding method for knockdown of a gene’s function. In Caenorhabditis elegans, feeding RNAi is most convenient, but this RNAi is not suitable for knockdown of multiple genes. Hence, we attempted to establish an efficient method of feeding RNAi for multiple knockdown. We produced bacteria yielding three distinct double-stranded RNAs bound to one another, and fed those bacteria to C. elegans. Quantitative RT-PCR and observation of phenotypes indicated that our method is much more efficient than the traditional one. Our method is useful for investigating genes’ functions in C. elegans.     

GENE SILENCING BY PARENTAL RNA INTERFERENCE IN THE GREEN RICE LEAFHOPPER,Nephotettix cincticeps (HEMIPTERA: CICADELLIDAE)

RNA interference (RNAi) has been widely used for investigating gene function in many nonmodel insect species. Parental RNAi causes gene knockdown in the next generation through the administration of double‐strand RNA (dsRNA) to the mother generation. In this study, we demonstrate that parental RNAi mediated gene silencing is effective in determining the gene function of the cuticle and the salivary glands in green rice leafhopper (GRH), Nephotettix cincticeps (Uhler). Injection of dsRNA of NcLac2 (9 ng/female) to female parents caused a strong knockdown of laccase‐2 gene of first instar nymphs, which eventually led to high mortality rates and depigmentation of side lines on the body. The effects of parental RNAi on the mortality of the nymphs were maintained through 12–14 days after the injections. We also confirmed the effectiveness of parental RNAi induced silencing on the gene expressed in the salivary gland, the gene product of which is passed from instar to instar. The parental RNAi method can be used to examine gene function by phenotyping many offspring nymphs with injection of dsRNA into a small number of parent females, and may be applicable to high‐efficiency determination of gene functions in this species.     

Interaction between differentiating cell- and niche-derived signals in hematopoietic progenitor maintenance

Maintenance of a hematopoietic progenitor population requires extensive interaction with cells within a microenvironment or niche. In the Drosophila hematopoietic organ, niche-derived Hedgehog signaling maintains the progenitor population. Here, we show that the hematopoietic progenitors also require a signal mediated by Adenosine deaminase growth factor A (Adgf-A) arising from differentiating cells that regulates extracellular levels of adenosine. The adenosine signal opposes the effects of Hedgehog signaling within the hematopoietic progenitor cells and the magnitude of the adenosine signal is kept in check by the level of Adgf-A secreted from differentiating cells. Our findings reveal signals arising from differentiating cells that are required for maintaining progenitor cell quiescence and that function with the niche-derived signal in maintaining the progenitor state. Similar homeostatic mechanisms are likely to be utilized in other systems that maintain relatively large numbers of progenitors that are not all in direct contact with the cells of the niche.     

Hematopoiesis at the onset of metamorphosis: terminal differentiation and dissociation of the <Emphasis Type="Italic">Drosophila</Emphasis> lymph gland

The Drosophila melanogaster hematopoietic organ, called lymph gland, proliferates and differentiates throughout the larval period. The lymph gland of the late larva is comprised of a large primary lobe and several smaller secondary lobes. Differentiation into two types of hemocytes, plasmatocytes and crystal cells, is confined to the outer layer (cortical zone) of the primary lobe; the center of the primary lobe (medullary zone), as well as the secondary lobes, contain only proliferating prohemocytes. A small cluster of cells located at the posterior tip of the primary lobe serves as a signaling center (PSC) that inhibits precocious differentiation of the medullary zone. The larval lymph gland is stabilized by layers of extracellular matrix (basement membranes) that surround individual hemocytes, groups of hemocytes, as well as the lymph gland as a whole. In this paper, we investigated the events shaping the lymph gland in the early pupa. The lymph gland dissociates and hemocytes disperse during the first 12 h after puparium formation (APF), leaving behind empty husks of basement membrane. Prior to lymph gland dissociation, cells of the medullary zone differentiate, expressing the early differentiation marker Peroxidasin (Pxn), as well as, in part, the late differentiation marker P1. Cells of the PSC spread throughout the pupal lymph gland prior to their dispersal. Cells of the secondary lobes undergo a rapid phase of proliferation that lasts until 8 h APF, followed by expression of Pxn and dispersal. These hemocytes do not express P1, indicating that they disperse prior to full maturation.