There and back again: The mechanisms of differentiation and transdifferentiation in Drosophila blood cells.

Transdifferentiation is a conversion of an already differentiated cell type into another cell type without the involvement of stem cells. This transition is well described in the case of vertebrate immune cells, as well as in Drosophila melanogaster, which therefore serves as a suitable model to study the process in detail. In the Drosophila larva, the latest single-cell sequencing methods enabled the clusterization of the phagocytic blood cells, the plasmatocytes, which are capable of transdifferentiation into encapsulating cells, the lamellocytes. Here we summarize the available data of the past years on the plasmatocyte-lamellocyte transition, and make an attempt to harmonize them with transcriptome-based blood cell clustering to better understand the underlying mechanisms of transdifferentiation in Drosophila, and in general.

Peeling Back the Layers of Lymph Gland Structure and Regulation.

During the past 60 years, the fruit fly, Drosophila melanogaster, has proven to be an excellent model to study the regulation of hematopoiesis. This is not only due to the evolutionarily conserved signalling pathways and transcription factors contributing to blood cell fate, but also to convergent evolution that led to functional similarities in distinct species. An example of convergence is the compartmentalization of blood cells, which ensures the quiescence of hematopoietic stem cells and allows for the rapid reaction of the immune system upon challenges. The lymph gland, a widely studied hematopoietic organ of the Drosophila larva, represents a microenvironment with similar features and functions to classical hematopoietic stem cell niches of vertebrates. Lymph gland studies were effectively supported by the unparalleled toolkit developed in Drosophila, which enabled the high-resolution investigation of the cellular composition and regulatory interaction networks of the lymph gland. In this review, we summarize how our understanding of lymph gland structure and hematopoietic cell-to-cell communication evolved during the past decades and compare their analogous features to those of the vertebrate hematopoietic stem cell niche.

Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster.

The blood cells, or hemocytes, in Drosophila participate in the immune response through the production of antimicrobial peptides, the phagocytosis of bacteria, and the encapsulation of larger foreign particles such as parasitic eggs; these immune reactions are mediated by phylogenetically conserved mechanisms. The encapsulation reaction is analogous to the formation of granuloma in vertebrates, and is mediated by large specialized cells, the lamellocytes. The origin of the lamellocytes has not been formally established, although it has been suggested that they are derived from the lymph gland, which is generally considered to be the main hematopoietic organ in the Drosophila larva. However, it was recently observed that a subepidermal population of sessile blood cells is released into the circulation in response to a parasitoid wasp infection. We set out to analyze this phenomenon systematically. As a result, we define the sessile hemocytes as a novel hematopoietic compartment, and the main source of lamellocytes.

A Novel Method for Primary Blood Cell Culturing and Selection in Drosophila melanogaster.

The blood cells of the fruit fly Drosophila melanogaster show many similarities to their vertebrate counterparts, both in their functions and their differentiation. In the past decades, a wide palette of immunological and transgenic tools and methods have been developed to study hematopoiesis in the Drosophila larva. However, the in vivo observation of blood cells is technically restricted by the limited transparency of the body and the difficulty in keeping the organism alive during imaging. Here we describe an improved ex vivo culturing method that allows effective visualization and selection of live blood cells in primary cultures derived from Drosophila larvae. Our results show that cultured hemocytes accurately represent morphological and functional changes following immune challenges and in case of genetic alterations. Since cell culturing has hugely contributed to the understanding of the physiological properties of vertebrate blood cells, this method provides a versatile tool for studying Drosophila hemocyte differentiation and functions ex vivo.

Immunoprofiling of Drosophila Hemocytes by Single-cell Mass Cytometry.

Single-cell mass cytometry (SCMC) combines features of traditional flow cytometry (i.e., fluorescence-activated cell sorting) with mass spectrometry, making it possible to measure several parameters at the single-cell level for a complex analysis of biological regulatory mechanisms. In this study, weoptimizedSCMC to analyze hemocytes of the Drosophila innate immune system. We used metal-conjugated antibodies (against cell surface antigens H2, H3, H18, L1, L4, and P1, and intracellular antigens 3A5 and L2) and anti-IgM (against cell surface antigen L6) to detect the levels of antigens, while anti-GFP was used to detect crystal cells in the immune-induced samples. We investigated the antigen expression profile of single cells and hemocyte populations in naive states, in immune-induced states, in tumorous mutants bearing a driver mutation in the Drosophila homologue of Janus kinase (hopTum) and carrying a deficiency of the tumor suppressor gene lethal(3)malignant blood neoplasm-1  [l(3)mbn1], as well as in stem cell maintenance-defective hdcΔ84 mutant larvae. Multidimensional analysis enabled the discrimination of the functionally different major hemocyte subsets for lamellocytes, plasmatocytes, and crystal cells, anddelineated the unique immunophenotype of Drosophila mutants. We have identified subpopulations of L2+/P1+ and L2+/L4+/P1+ transitional phenotype cells in the tumorous strains l(3)mbn1 and hopTum, respectively, and a subpopulation of L4+/P1+ cells upon immune induction. Our results demonstrated for the first time that SCMC, combined with multidimensional bioinformatic analysis, represents a versatile and powerful tool to deeply analyze the regulation of cell-mediated immunity of Drosophila.

Regression plane concept for analysing continuous cellular processes with machine learning.

Biological processes are inherently continuous, and the chance of phenotypic discovery is significantly restricted by discretising them. Using multi-parametric active regression we introduce the Regression Plane (RP), a user-friendly discovery tool enabling class-free phenotypic supervised machine learning, to describe and explore biological data in a continuous manner. First, we compare traditional classification with regression in a simulated experimental setup. Second, we use our framework to identify genes involved in regulating triglyceride levels in human cells. Subsequently, we analyse a time-lapse dataset on mitosis to demonstrate that the proposed methodology is capable of modelling complex processes at infinite resolution. Finally, we show that hemocyte differentiation in Drosophila melanogaster has continuous characteristics.

Identification of reference markers for characterizing honey bee (Apis mellifera) hemocyte classes.

Cell mediated immunity of the honey bee (Apis mellifera) involves the activity of several hemocyte populations, currently defined by morphological features and lectin binding characteristics. The objective of the present study was to identify molecular markers capable of characterizing subsets of honey bee hemocytes. We developed and employed monoclonal antibodies with restricted reactions to functionally distinct hemocyte subpopulations. Melanizing cells, known as oenocytoids, were defined by an antibody to prophenoloxidase, aggregating cells were identified by the expression of Hemolectin, and phagocytic cells were identified by a marker expressed on granulocytes. We anticipate that this combination of antibodies not only allows for the detection of functionally distinct hemocyte subtypes, but will help to further the exploration of hematopoietic compartments, as well as reveal details of the honey bee cellular immune defense against parasites and microbes.

Headcase is a Repressor of Lamellocyte Fate in Drosophila melanogaster.

Due to the evolutionary conservation of the regulation of hematopoiesis, Drosophila provides an excellent model organism to study blood cell differentiation and hematopoietic stem cell (HSC) maintenance. The larvae of Drosophila melanogaster respond to immune induction with the production of special effector blood cells, the lamellocytes, which encapsulate and subsequently kill the invader. Lamellocytes differentiate as a result of a concerted action of all three hematopoietic compartments of the larva: the lymph gland, the circulating hemocytes, and the sessile tissue. Within the lymph gland, the communication of the functional zones, the maintenance of HSC fate, and the differentiation of effector blood cells are regulated by a complex network of signaling pathways. Applying gene conversion, mutational analysis, and a candidate based genetic interaction screen, we investigated the role of Headcase (Hdc), the homolog of the tumor suppressor HECA in the hematopoiesis of Drosophila. We found that naive loss-of-function hdc mutant larvae produce lamellocytes, showing that Hdc has a repressive role in effector blood cell differentiation. We demonstrate that hdc genetically interacts with the Hedgehog and the Decapentaplegic pathways in the hematopoietic niche of the lymph gland. By adding further details to the model of blood cell fate regulation in the lymph gland of the larva, our findings contribute to the better understanding of HSC maintenance.

Genes encoding cuticular proteins are components of the Nimrod gene cluster in Drosophila.

The Nimrod gene cluster, located on the second chromosome of Drosophila melanogaster, is the largest synthenic unit of the Drosophila genome. Nimrod genes show blood cell specific expression and code for phagocytosis receptors that play a major role in fruit fly innate immune functions. We previously identified three homologous genes (vajk-1, vajk-2 and vajk-3) located within the Nimrod cluster, which are unrelated to the Nimrod genes, but are homologous to a fourth gene (vajk-4) located outside the cluster. Here we show that, unlike the Nimrod candidates, the Vajk proteins are expressed in cuticular structures of the late embryo and the late pupa, indicating that they contribute to cuticular barrier functions.

Characterization of the grappa gene, the Drosophila histone H3 lysine 79 methyltransferase.

We have identified a novel gene named grappa (gpp) that is the Drosophila ortholog of the Saccharomyces cerevisiae gene Dot1, a histone methyltransferase that modifies the lysine (K)79 residue of histone H3. gpp is an essential gene identified in a genetic screen for dominant suppressors of pairing-dependent silencing, a Polycomb-group (Pc-G)-mediated silencing mechanism necessary for the maintenance phase of Bithorax complex (BX-C) expression. Surprisingly, gpp mutants not only exhibit Pc-G phenotypes, but also display phenotypes characteristic of trithorax-group mutants. Mutations in gpp also disrupt telomeric silencing but do not affect centric heterochromatin. These apparent contradictory phenotypes may result from loss of gpp activity in mutants at sites of both active and inactive chromatin domains. Unlike the early histone H3 K4 and K9 methylation patterns, the appearance of methylated K79 during embryogenesis coincides with the maintenance phase of BX-C expression, suggesting that there is a unique role for this chromatin modification in development.

The raspberry Gene Is Involved in the Regulation of the Cellular Immune Response in Drosophila melanogaster.

Drosophila is an extremely useful model organism for understanding how innate immune mechanisms defend against microbes and parasitoids. Large foreign objects trigger a potent cellular immune response in Drosophila larva. In the case of endoparasitoid wasp eggs, this response includes hemocyte proliferation, lamellocyte differentiation and eventual encapsulation of the egg. The encapsulation reaction involves the attachment and spreading of hemocytes around the egg, which requires cytoskeletal rearrangements, changes in adhesion properties and cell shape, as well as melanization of the capsule. Guanine nucleotide metabolism has an essential role in the regulation of pathways necessary for this encapsulation response. Here, we show that the Drosophila inosine 5'-monophosphate dehydrogenase (IMPDH), encoded by raspberry (ras), is centrally important for a proper cellular immune response against eggs from the parasitoid wasp Leptopilina boulardi. Notably, hemocyte attachment to the egg and subsequent melanization of the capsule are deficient in hypomorphic ras mutant larvae, which results in a compromised cellular immune response and increased survival of the parasitoid.

The Nimrod transmembrane receptor Eater is required for hemocyte attachment to the sessile compartment in Drosophila melanogaster.

Eater is an EGF-like repeat transmembrane receptor of the Nimrod family and is expressed in Drosophila hemocytes. Eater was initially identified for its role in phagocytosis of both Gram-positive and Gram-negative bacteria. We have deleted eater and show that it appears to be required for efficient phagocytosis of Gram-positive but not Gram-negative bacteria. However, the most striking phenotype of eater deficient larvae is the near absence of sessile hemocytes, both plasmatocyte and crystal cell types. The eater deletion is the first loss of function mutation identified that causes absence of the sessile hemocyte state. Our study shows that Eater is required cell-autonomously in plasmatocytes for sessility. However, the presence of crystal cells in the sessile compartment requires Eater in plasmatocytes. We also show that eater deficient hemocytes exhibit a cell adhesion defect. Collectively, our data uncovers a new requirement of Eater in enabling hemocyte attachment at the sessile compartment and points to a possible role of Nimrod family members in hemocyte adhesion.

Multinucleated Giant Hemocytes Are Effector Cells in Cell-Mediated Immune Responses of Drosophila.

We identified and characterized a so far unrecognized cell type, dubbed the multinucleated giant hemocyte (MGH), in the ananassae subgroup of Drosophilidae. Here, we describe the functional and ultrastructural characteristics of this novel blood cell type as well as its characterization with a set of discriminative immunological markers. MGHs are encapsulating cells that isolate and kill the parasite without melanization. They share some properties with but differ considerably from lamellocytes, the encapsulating cells of Drosophila melanogaster, the broadly used model organism in studies of innate immunity. MGHs are nonproliferative effector cells that are derived from phagocytic cells of the sessile tissue and the circulation, but do not exhibit phagocytic activity. In contrast to lamellocytes, MGHs are gigantic cells with filamentous projections and contain many nuclei, which are the result of the fusion of several cells. Although the structure of lamellocytes and MGHs differ remarkably, their function in the elimination of parasites is similar, which is potentially the result of the convergent evolution of interactions between hosts and parasites in different geographic regions. MGHs are highly motile and share several features with mammalian multinucleated giant cells, a syncytium of macrophages formed during granulomatous inflammation.

The cell-mediated immunity of Drosophila melanogaster: hemocyte lineages, immune compartments, microanatomy and regulation.

In the animal kingdom, innate immunity is the first line of defense against invading pathogens. The dangers of microbial and parasitic attacks are countered by similar mechanisms, involving the prototypes of the cell-mediated immune responses, the phagocytosis and encapsulation. Work on Drosophila has played an important role in promoting an understanding of the basic mechanisms of phylogenetically conserved modules of innate immunity. The aim of this review is to survey the developments in the identification and functional definition of immune cell types and the immunological compartments of Drosophila melanogaster. We focus on the molecular and developmental aspects of the blood cell types and compartments, as well as the dynamics of blood cell development and the immune response. Further advances in the characterization of the innate immune mechanisms in Drosophila will provide basic clues to the understanding of the importance of the evolutionary conserved mechanisms of innate immune defenses in the animal kingdom.

In vivo immunostaining of hemocyte compartments in Drosophila for live imaging.

In recent years, Drosophila melanogaster has become an attractive model organism in which to study the structure and development of the cellular immune components. The emergence of immunological markers greatly accelerated the identification of the immune cells (hemocytes), while the creation of genetic reporter constructs allowed unique insight into the structural organization of hematopoietic tissues. However, investigation of the hemocyte compartments by the means of immunological markers requires dissection and fixation, which regularly disrupt the delicate structure and hamper the microanatomical characterization. Moreover, the investigation of transgenic reporters alone can be misleading as their expression often differs from the native expression pattern of their respective genes. We describe here a method that combines the reporter constructs and the immunological tools in live imaging, thereby allowing use of the array of available immunological markers while retaining the structural integrity of the hematopoietic compartments. The procedure allows the reversible immobilization of Drosophila larvae for high-resolution confocal imaging and the time-lapse video analysis of in vivo reporters. When combined with our antibody injection-based in situ immunostaining assay, the resulting double labeling of the hemocyte compartments can provide new information on the microanatomy and functional properties of the hematopoietic tissues in an intact state. Although this method was developed to study the immune system of Drosophila melanogaster, we anticipate that such a combination of genetic and immunological markers could become a versatile technique for in vivo studies in other biological systems too.

A novel method for the identification of factors involved in host-pathogen interactions in Drosophila melanogaster.

A new method was established, standardized and validated for screening factors involved in the response to septic injury in Drosophila melanogaster. The method, based on inducing lesion by removing the tarsal segments of the first pair of legs of Drosophila adults and exposing them to different bacteria, imitates injury that often occurs in the natural habitat. The method is easy to perform, highly reproducible and suitable for large-scale genetic screens with the aim of identifying factors involved in host-pathogen interactions. The technique was validated by using mutant variations of different components of the immune response, blood clotting as well as the involvement of a number of genes known to be instrumental in the humoral and cell-mediated immune responses of Drosophila was confirmed. Moreover, the combination of the present method with antibiotic treatment allows the screening of potential antimicrobial drugs in vivo.

Variation of NimC1 expression in Drosophila stocks and transgenic strains.

The NimC1 molecule has been described as a phagocytosis receptor, and is being used as a marker for professional phagocytes, the plasmatocytes, in Drosophila melanogaster. In studies including tumor-biology, developmental biology, and cell mediated immunity, monoclonal antibodies (P1a and P1b) to the NimC1 antigen are used. As we observed that these antibodies did not react with plasmatocytes of several strains and genetic combinations, a molecular analysis was performed on the structure of the nimC1 gene. In these strains we found 2 deletions and an insertion within the nimC1 gene, which may result in the production of a truncated NimC1 protein. The NimC1 positivity was regained by recombining the mutation with a wild-type allele or by using nimC1 mutant lines under heterozygous conditions. By means of these procedures or using the recombined stock, NimC1 can be used as a marker for phagocytic cells in the majority of the possible genetic backgrounds.

Cell lineage tracing reveals the plasticity of the hemocyte lineages and of the hematopoietic compartments in Drosophila melanogaster.

Much of our knowledge on hematopoiesis, hematopoietic compartments, hematopoietic cell lineages and immunity has been derived from studies on the vertebrate immune system. The sophisticated innate immunity of insects, the phylogenetic conservation and the power of Drosophila genetics allowed the investigation of immune cell (hemocyte) lineage relationships in Drosophila melanogaster. The development of the hemocyte lineages in Drosophila is a result of a precisely regulated succession of intracellular and intercellular events, though the nature and extent of these interactions are not known. We describe here a cell lineage tracing system set up to analyze the development of hemocyte lineages and functionally distinct hemocyte subsets. This system allowed us to distinguish two major embryonic hemocyte lineages, the crq and Dot lineages, in two, physically separated compartments, the embryonic macrophages and the embryonic lymph gland. We followed the fate and development of these lineages in the construction of the larval hematopoietic compartments and during the cell-mediated immune response, the encapsulation reaction. Our results revealed the considerable plasticity and concerted action of the hematopoietic compartments and the hemocyte lineages in the development of the innate immune system and in the course of the cell-mediated immune response in Drosophila.

APOE epsilon status in Hungarian patients with primary progressive multiple sclerosis.

PRINCIPLES: Apolipoprotein E (ApoE), an important glycoprotein in the transport, uptake and redistribution of cholesterol, is necessary in nerve tissue repair. The APOE gene (APOE) is involved in neurodegenerative diseases, the best-known association being that between the APOE ε4 allele and Alzheimer's disease. Multiple sclerosis (MS) is a chronic inflammatory neurological disease. The aim of this study was to assess (multicentre assessment) the possible influence of the APOE gene on the susceptibility of primary progressive MS (PPMS) in Hungary. METHODS: Polymerase chain reaction and restriction fragment length polymorphism were carried out on DNA isolated from 135 volunteers. RESULTS: The number of PPMS patients without the ε2 allele was found to be remarkably high, whilst the ε2 allele was overrepresented in the RRMS group. A markedly high frequency of the ε4 allele was found in the PPMS group and a very low frequency in the HC group. With regards to the clinical parameters, significant differences were observed between the RRMS and PPMS groups. Differences were also detected regarding the EDSS and MSSS scores when the patients were grouped by the presence or absence of the ε2 allele. All of the observed differences in the clinical parameters disappeared when the patients were further stratified by the type of MS. CONCLUSIONS: Our findings suggest that the presence of the ε2 and ε4 alleles may play a role in the development of the disease. However, if any type of the disease has already developed the alleles show no association with the clinical parameters.