Activation Of The Cellular Immune Response In

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Activation of the Cellular Immune Response in Drosophila melanogaster Larvae Ines Anderl Department of Molecular Biology Umeå University, Umeå, Sweden 2015 Responsible publisher under swedish law: the Dean of the Medical Faculty This work is protected by the Swedish Copyright Legislation (Act 1960:729) ISBN: 978-91-7601-317-5 ISSN: 0346-6612-1741 Picture top left: Drosophila blood cells; top right: encapsulated wasp egg; bottom: wasp-infected Drosophila larva; back cover: plasmatocytes (green) and lamellocytes (red) Electronic version available at http://umu.diva-portal.org/ Printed by: Print & Media, Umeå University Umeå, Sweden 2015 “Time flies like an arrow; fruit flies like a banana.” Groucho Marx Table of Contents Table of Contents............................................................. i   Abstract ......................................................................... iii   Papers included in the thesis ......................................... iv   Background ..................................................................... 1   Introduction ................................................................................... 1   Drosophila blood cells ................................................................... 2   Drosophila hematopoiesis ............................................................. 3   Lymph  gland  hematopoiesis  ..............................................................................  3   Peripheral  hematopoiesis  ...................................................................................  4   Adult  hematopoiesis  ..............................................................................................  6   Immune response to parasitoid wasps .......................................... 7   Drosophila as a research tool ........................................................ 8   Genetic Screens in Drosophila with focus on hemocytes ............. 9   Methods for hemocyte research .................................................. 10   Aims of the thesis .......................................................... 13   Article I ......................................................................... 14   “A directed screen for genes involved in Drosophila blood cell activation.” ........................................................................................ 14   Preface .......................................................................................... 14   Summary of main results............................................................. 14   Discussion .................................................................................... 14   Article II ........................................................................ 16   “Infection-induced proliferation is a central hallmark of the activation of the cellular immune response in Drosophila larvae.” 16   Preface and study design ............................................................. 16   Summary of main results............................................................. 17   Discussion .................................................................................... 17   Article III....................................................................... 21   “Genetic screen in Drosophila larvae links ird1 function to Toll signaling in the fat body and hemocyte motility.” ........................... 21   Preface .......................................................................................... 21   Summary of main results............................................................. 21   Discussion .................................................................................... 21   Article IV ...................................................................... 24   “Control of Drosophila blood cell activation via Toll signaling in the fat body.” ..................................................................................... 24   Preface .......................................................................................... 24   Summary of main results............................................................. 24   i Discussion .................................................................................... 24   Acknowledgements .......................................................27   References ................................................................... 28   ii Abstract During the last 40 years, Drosophila melanogaster has become an invaluable tool in understanding innate immunity. The innate immune system of Drosophila consists of a humoral and a cellular component. While many details are known about the humoral immune system, our knowledge about the cellular immune system is comparatively small. Blood cells or hemocytes constitute the cellular immune system. Three blood types have been described for Drosophila larvae. Plasmatocytes are phagocytes with a plethora of functions. Crystal cells mediate melanization and contribute to wound healing. Plasmatocytes and crystal cells constitute the blood cell repertoire of a healthy larva, whereas lamellocytes are induced in a demand-adapted manner after infection with parasitoid wasp eggs. They are involved in the melanotic encapsulation response against parasites and form melanotic nodules that are also referred to as tumors. In my thesis, I focused on unraveling the mechanisms of how the immune system orchestrates the cellular immune response. In particular, I was interested in the hematopoiesis of lamellocytes. In Article I, we were able to show that ectopic expression of key components of a number of signaling pathways in blood cells induced the development of lamellocytes, led to a proliferative response of plasmatocytes, or to a combination of lamellocyte activation and plasmatocyte proliferation. In Article II, I combined newly developed fluorescent enhancer-reporter constructs specific for plasmatocytes and lamellocytes and developed a “dual reporter system” that was used in live microscopy of fly larvae. In addition, we established flow cytometry as a tool to count total blood cell numbers and to distinguish between different blood cell types. The “dual reporter system” enabled us to differentiate between six blood cell types and established proliferation as a central feature of the cellular immune response. The combination flow cytometry and live imaging increased our understanding of the tempo-spatial events leading to the cellular immune reaction. In Article III, I developed a genetic modifier screen to find genes involved in the hematopoiesis of lamellocytes. I took advantage of the gain-of-function phenotype of the Tl10b mutation characterized by an activated cellular immune system, which induced the formation blood cell tumors. We screened the right arm of chromosome 3 for enhancers and suppressors of this mutation and uncovered ird1. Finally in Article IV, we showed that the activity of the Toll signaling pathway in the fat body, the homolog of the liver, is necessary to activate the cellular immune system and induce lamellocyte hematopoiesis. iii Papers included in the thesis This thesis is based on the following publications, which will be referred to by roman numerals (I - IV). All publications are reproduced with the permission from the publishers. Article I Zettervall, C.J., Anderl, I., Williams, M.J., Palmer, R., Kurucz, E., Ando, I., and Hultmark, D. (2004). A directed screen for genes involved in Drosophila blood cell activation. Proc. Natl. Acad. Sci. USA 101, 14192-14197. Article II Anderl, I., Vesala, L., Ihalainen, T., Vanha-aho, L.-M., Rämet, M., and Hultmark, D. (2015). Infection-induced proliferation is a central hallmark of the activation of the cellular immunes response in Drosophila larvae. (Manuscript, I. Anderl and L. Vesala share first authorship) Article III Schmid, M.R., Anderl, I., Vesala, L., Vo, H., Valanne, S., Yang, H., Kronhamn, J., Rämet, M., Rusten, T.E., Hultmark, D. (2015). Genetic screen in Drosophila larvae links ird1 function to Toll signaling in the fat body and hemocyte motility. (Manuscript) Article IV Schmid, M.R., Anderl, I., Vesala, L., Vanha-aho, L.-M., Deng, X.-J., Rämet, M., and Hultmark, D. (2014). Control of Drosophila blood cell activation via Toll signaling in the fat body. PLoS One 9, e102568. (M. Schmid and I. Anderl share first authorship.) iv Background Introduction Every organism, regardless of size and complexity, must protect itself against assaults from non-self to “live long and prosper”. The discipline of immunology was founded by Elie Metchnikoff and Paul Ehrlich who were awarded the Nobel Prize in 1908 “in recognition for their work on immunity” Metchnikoff´s discovery of phagocytosis spawned innate immunity and Ehrlich´s side-chain theory adaptive immunity [1]. The first frontline to infection are physical barriers. The skin protects the outside of our bodies and mucosal tissues, such as lung, airways, and gut the inside. Mucous tissues secrete fluids like tears, saliva, or mucus that contain compounds that restrict the growth of microorganisms. Moreover, mucosal tissues are colonized by immune cells. Two forms of immunity exist - the innate and the adaptive immunity. Innate immunity is fast and general and has a cellular and humoral component. Pattern recognition receptors (PRRs) expressed by innate immune cells recognize pathogenassociated molecular patterns (PAMPs) on the surface of intruders [2], [3] and phagocytize them. In mammals, the complement system constitutes the humoral arm of innate immunity. Their adaptive immunity comprises B and T lymphocytes, is highly specific, and confers immunological memory. Autoreactive B and T cells are eliminated during their development. The innate and adaptive immune system cooperate: “There is no antigen-specific immunity without “instruction” by the innate immune system and no efficacious innate immune response without the guidance of acquired immunity” [1]. Mammalian blood cells are born in primary lymphoid tissues, the bone marrow and the thymus. They develop form multipotent hematopoietic stem cells by hierarchical lineage restriction (reviewed in [4]). This view has recently been challenged by the heterogeneity of tissue macrophages (reviewed in [5]). The adaptive immune response is generated in the secondary lymphoid tissues, the lymph nodes, the spleen, and the mucosa-associated lymphoid tissue (MALT). In the genomic era much effort has been dedicated to elucidating the origin of the immune system. It has become clear that the immune system evolved in cooperation with the commensal microbiota [6]. Notably, our gut microbiota shape our adaptive immune system (reviewed in [7]). And also in the fly, the intestinal homeobox gene Caudal represses the Toll-dependent expression of antimicrobial peptides. If this regulation is abrogated, the commensal flora of the fly gut becomes unbalanced resulting in the death of the animal [8]. Vertebrates, which comprise only a small group within the animal kingdom, are so far the only group of animals known to have adaptive immunity, but innate immunity is ubiquitious. From an evolutionary perspective, insects are the most successful class in the animal kingdom representing approximately half of all existing animal species [9], however, they only have an innate immune system. It has been argued that the advantage of the specificity of the immune reaction conferred by adaptive immunity might be in saving energy resources [10]. The innate immune system of insects consists of a humoral and cellular arm. The fat body, the homolog of the liver, is the central organ in secreting humoral components. Blood cells or hemocytes are the effector cells of the cellular arm of the immune system. Drosophila melanogaster has become the state of the art model organism for innate immunity research during the last 40 years (reviewed in [11]). Work on the humoral immune system of the cecropia moth led to the discovery of antimicrobial peptides that are secreted by the fat body into the hemolymph in response to bacterial or fungal infection (reviewed in [12]). In 1972, Bertil Rasmusons lab showed for the first time that antimicrobial activity is inducible in Drosophila 1 [13]. Ingrid Faye´s group reported in 1992 that in the cecropia moth a nuclear immuneresponsive factor binds to NFκB-like motifs [14]. In 1993 the Engström, Faye, and Hultmark labs jointly reported for the first time that κB-like motifs regulate the induction of immune genes in Drosophila [15]. And in 1995 the Hultmark lab showed that Toll signaling is able to introduce Cecropin gene expression [16]. In 1996 Bruno Lemaitre in the Hoffmann lab reported that Micrococcus-induced Drosomycin expression is dependent on Toll signaling [17]. These and other findings spawned a renewed interest in innate immunity (reviewed in [11]). Jules Hoffman and Bruce Beutler were jointly awarded the Nobel Prize for Physiology and Medicine in 2011 “for their discoveries concerning the activation of innate immunity” Analogous to our skin, the cuticle of the exoskeleton of insects is the first physical barrier to infection. But in contrast to vertebrates, insects have an open circulatory system, which needs quick repair when injured to prevent bleeding to death. Therefore, the hemolymph harbors an efficient clotting or coagulation system to minimize bleeding and to prevent invading bacteria from spreading through the hemolymph [18], [19]. A quick and efficient wound healing response takes care of tissue repair [20], [21]. Wound healing and hemolymph clotting are complemented by melanization. Phenol oxidases (POs) induce the rapid polymerization of quinones that result in the production of the black-brown pigment melanin at the site of injury. Melanin traps bacteria in nodules and contributes to clot formation [22]. Melanin itself and byproducts of the melanization cascade possess antimicrobial activity [23], [22]. Finally, encapsulation is an immune response dedicated to the killing of parasitoids that are too big for phagocytosis like the eggs of parasitoid wasps (reviewed in [24], [25]). Melanization and encapsulation are immune reactions specific to arthropods (reviewed in [19], [26]). Blood cells constitute the cellular arm of innate immunity. This thesis is dedicated to hemocytes, specifically to lamellocytes. Therefore I primarily focus on the development and function of peripheral blood cells in the larva. Drosophila blood cells The cellular immune system of insects is represented by blood cells or hemocytes. As insects do not have adaptive immunity, their hemocytes bear resemblance to mammalian blood cells of the myloid lineage (reviewed in [27]). In Drosophila melanogaster three different hemocyte types can be found: plasmatocytes, crystal cells, and lamellocytes. Plasmatocytes and crystal cells are present in all developmental stages of the fruit fly (reviewed in [28]), whereas lamellocytes are induced only in response to infection by pathogens to large to phagocytize (reviewed in [25]). In response to parasitoid wasp infection, lamellocyte numbers rise swiftly to encapsulate and thereby kill the intruder [29]. In unchallenged and healthy animals, plasmatocytes are the most numerous cell type constituting 90 to 95 % of all blood cells [30], [31]. They are small spherical cells with a diameter up to 12 μm [30], [32]. Plasmatocytes are the workhorse of the cellular immune systems with various functions in immunity and development. They phagocytize apoptotic tissue and bacteria (reviewed in for example [28]), participate in the encapsulation response (reviewed in [25]), patrol the hemolymph as sentinels [33], synthesize antimicrobial peptides [34], cytokines [35], [36], [37], [38], [39], and extracellular matrix [40], [41], [42], transdifferentiate into crystal cells [43] and lamellocytes [44], [45], [46], are involved in the priming of the immune response against bacteria [47], contribute to tissue remodeling [48], [35], regeneration, and homeostatis [49], [50], [39]. Despite the plethora of functions, plasmatocyte depletion in larvae is not necessary for survival [51]. The crystals within the cytoplasm of crystal cells were the inspiration for the naming of this cell type. They are spherical, slightly bigger than plasmatocytes, and have a golden hue in phase contrast microscopy. Crystal cells are dedicated to 2 melanization upon wounding and bacterial infection [52]. At the wound site, they contribute to scab formation [20]. Crystal cells express two of the three PPO (prophenol oxidase) genes of the Drosophila melanogaster genome. PPO1 (CG42639) is quickly released into the hemolymph after wounding. PPO2 (CG8913) is stored within the crystals and only released after crystal cell rupture [52]. PPOrelease after crystal cells rapture is controlled by the Jun kinase basket (bsk), RhoGTPases, and the Drosophila TNF homolog eiger (egr) [53]. PPO1 and PPO2 require proteolytic cleavage for their activation [54]. PPO1 is cleaved by the CLIP serine protease Hayan [55]. The third PPO encoded in the Drosophila genome is PPO3 (CG42640). PPO3 is lamellocyte-specific, does not require proteolytic cleavage for activation, and is not sufficient to melanize encapsulated wasp eggs in a double mutant background of PPO1 and PPo2 [52]. Lamellocytes are large discoid cells that are only 1 μm thick [30], [32], [31]. They are effector cells only produced by the immune system when parasitoid wasps have deposited their eggs inside the Drosophila host [56], or at aberrant conditions, when they participate in forming melanotic nodules (reviewed in [57]). Drosophila hematopoiesis Hematopoiesis between Drosophila and vertebrates is evolutionary conserved [27]. Blood cells hematopoiesis occurs in waves in the embryo [27], in the larva [58], and in the adult fly [59]. In the Drosophila embryo, plasmatocytes and crystal cells are born and proliferate in the head mesoderm. Crystal cells never leave this site during embryonic development, whereas plasmatocytes migrate through the embryo as macrophages and phagocytize apoptotic tissue [60], [61]. The embryonic macrophages colonize the hemocoel of the Drosophila larva as peripheral hemocytes and persist till the adult fly [62]. In the larval hemocoel, peripheral blood cells either circulate or are sessile [31]. The sessile hemocyte compartment is now regarded as a hematopoietic niche [63], [64], [43]. The lymph gland arises from the thoracic mesoderm [62]. The lymph glands have long since been regarded the hematopoietic organ of the larva [65]. They grow throughout larval development and disintegrate at the beginning of metamorphosis [66]. Cells derived from the secondary lymph gland lobes home to hematopoitic clusters in the abdomen of the fly [59]. Lymph gland hematopoiesis During the last 15 years, the lymph glands have become an excellent model for hematopoiesis. The lymph glands consist of several paired lobes adjacent to the heart (Figure 1). The primary lymph gland lobes consist of distinct zones. Quiescent hematopoietic stem cells, the prohemocytes, reside in the medullary zone (MZ), and differentiated hemocytes are found in the cortical zone (CZ). The posterior signaling center (PSC) constitutes the hematopoietic niche that controls the balance between the prohemocytes and differentiating hemocytes in the medullary zone of the lymph gland (Figure 1) [67]. 3 CZ MZ PSC 2nd lobes crystal cell plasmatocyte PSC cell prohemocyte pericardial cell Figure 1 Mature lymph gland (CZ – Cortical Zone, MZ – Medullary Zone, PSC – Posterior Signaling Center) Peripheral hematopoiesis It has previously been assumed that lymph glands are the hematopoietic organ that gives rise to all larval blood cells [27]. However, transplantation experiments from the hemocyte anlage at the blastoderm stage revealed that embryonic plasmatocytes are the founder population of peripheral larval hemocytes and that they persist to the imagines [62]. Two peripheral larval blood cell populations have been described, the circulating and the sessile cells (Figure 2). Circulating hemocytes mostly consist of plasmatocytes. Depending on genetic background, they are estimated to account for 90 to 95 % and crystal cells for the remaining 5 to 10 % [30]). Also a low number of prohemocytes was observed in circulation [32], [31], [63]. Circulating hemocyte numbers during the larval stage increase from fewer than 200 at the beginning of the first instar to more than 6000 cells at the end of the third larval instar [30], [31], [64]. Sessile hemocytes account for approximately the same number as circulating cells because in the eater1 mutant, where sessile cells are absent, circulating cell numbers double in comparison to wildtype controls [68]. Whereas Lanot et al. estimated that at least one third of the blood cells are attached to the integument [31]. Circulating hemocytes are regarded as sentinels that passively patrol the hemocoel propelled by the hemolymph flow. During wound healing, they are recruited by “direct capture“ from the hemolymph [33]. Sessile cells attach to larval tissues and comprise plasmatocytes and crystal cells (Figure 1A). Primarily they adhere to the dorsal integument in a repeated segmented pattern [31], [69], [70] where they reside and proliferate in-between the epidermis and muscles tightly associated with neurons of the peripheral nervous system. The peripheral neurons are thought to provide a trophic environment of yet unknown growth factors, and therefore these epidermal-muscular pockets are regarded as a hematopoietic niche for circulating and sessile hemocytes [64]. When embryonic hemocytes first colonize the larval hemocoel, they accumulate on the integument of the two posteriormost abdominal segments. In later larval stages, the density of 4 A Subepithelial hemocyte patches Lymph glands Posterior hematopoietic tissue (PHT) non-infected Plasmatocytes Crystal cells B Secondary lymph gland lobes Encapsulated parasite eggs infected Plasmatocytes Lamellocytes Figure 2 Hemocytes and hematopoietic compartments in the Drosophila larva In (A) non-infected and (B) wasp-infected animals (published by Anderl & Hultmark 2015 [71] and presented here with small modifications) sessile cells is highest in this part of the larva [32], [72]. It has been suggested to refer to these cells as the posterior hematopoietic tissue (PHT) [72]. In addition, hemocytes can also be found adhering to imaginal discs [72], [62] and elsewhere. Blood cells cycle between sessile and circulating states [33], [73], [64]. The expression of the Nimrod receptor eater in plasmatocytes mediates the adhesion to the sessile compartment, however the nature of the eater ligand is still elusive [68]. It is possible that eater binds to a component of the basement membrane, as it was shown that Kc167 cells bind to lamin in a syndecan-dependent manner and hemocytes adhere to lamin in vivo [74]. Mature plasmatocytes proliferate by self-renewel in circulation [30], [64] and in the epidermal-muscular pockets where their proliferation rate is higher [64]. Plasmatocytes express croquemort (crq), Peroxidasin (Pxn), eater (reviewed in [75], and u-shaped (ush) [76], [45]. Misexpression of Pvf2 [77], Egfr, and Ras85D [70] in hemocytes induce proliferation of plasmatocytes. Crystal cell numbers also increase during larval stages. Paradoxically, all reports agreed so far that mature crystal cells do not divide [30], [31]. Instead, plasmatocytes transdifferentiate into crystal cells in the sessile compartment in a Notch-Serrate dependent process that requires cell-to cell contacts [43], [78]. However, in eater1 mutants crystal cells do develop. Clumps of crystal cells and lamellocytes are observed in tight association in circulation [68]. This suggests that hemocyte-tohemocyte contacts are more important than growth factors from the hematopoietic niche. The enormous plasticity of the cellular immune system becomes evident during immune reactions to wasp infection or at aberrant genetic conditions that lead to socalled melanotic nodules. A new blood cell type, the lamellocyte, is generated. 5 Lamellocytes are not present in healthy Drosophila larvae. Therefore, they can only be studied when the larva is immunocompromised. It has been debated whether lamellocytes develop only in the lymph glands from prohemocytes and are released into circulation, or if mature plasmatocytes of the peripheral hemocyte population can transdifferentiate into lamellocytes. Rizki regarded lamellocytes as a specialized type of plasmatocyte that develops in circulation by flattening via a transitional cell type called podocyte [30], [79]. After wasp infection, circulating cell counts increase, and it was therefore assumed that sessile hemocytes detach and develop into lamellocytes [70]. In contrast, Meister and coworkers provided evidence that lamellocytes develop in the lymph glands from prohemocytes and are released into circulation, while not entirely excluding that lamellocytes can develop in the periphery from prohemocytes [31]. The hypothesis in favor of lamellocyte development in the lymph glands is corroborated by the observation that the primary lymph gland lobes of some parasitized larvae disperse [80] and by the complete absence of lamellocytes in a knot (collier) mutant where knot is acting in the PSC to control lamellocyte differentiation [81]. These hypotheses were tested, and further investigations established that after parasitization plasmatocytes and presumably prohemocytes, termed “double negatives” for not expressing NimC1 (plasmatocyte antigen) or L1 (lamellocyte antigen), left the sessile compartment. Lamellocytes appeared in circulation while the primary lymph gland lobes were still intact. In an infected larva, where the anterior half that contains the lymph glands was physically separated by ligation from the posterior half where most of the sessile hemocytes reside, lamellocytes developed only in the posterior ends of the larva [63]. Three independent lineage tracing studies confirmed that peripheral plasmatocytes indeed differentiate into lamellocytes. Ando and coworkers used two different drivers that are active in either the periphery (crqGAL4) or the lymph glands (Dot-GAL4). In non-infected larvae, the crq-lineage derived plasmatocytes were only found in circulation, whereas Dot-lineage cells resided only in the lymph glands. After parasitzation, lamellocytes in circulation originated from both the lymph glands and the peripheral hemocyte compartments [44]. Badenhorst and coworkers lineage traced the origin of lamellocytes after wasp infection to plasmatocytes [46]. Avet-Rochex et al. knocked down ush in embryonic plasmatocytes and were also able to show that plasmatocytes gave rise to lamellocytes [45]. The presence of melanotic nodules in the absence of wasp infection in Drosophila larvae has long since been associated with lamellocytes [82]. Mutations in genes of two well-known pathways involved in immunity, the Toll pathway and the Jak/Stat pathway, induce the formation of melanotic nodules [83], [84]. Two genetic screens, a misexpression screen [85] and an in vivo RNAi screen [45] aimed to find new genes involved in lamellocyte hematopoiesis. Lamellocyte hematopoiesis can be induced genetically by overactivation of genes such as in the Toll10b (Tl10b) mutation [83], [70], or by loss-of-function mutations in genes that repress lamellocyte hematopoiesis such such as ush [76], [45]. Moreover, lamellocyte formation is also controlled by epigenetic factors [85], [57]. It is noteworthy that melanotic nodules are not always associated with blood cells [86]. Adult hematopoiesis Adult hemocytes consist of plasmatocytes and crystal cells. They are able to phagocytose bacteria, but their cell number declines with age [87], [88]. It has been generally accepted that the adult fly does not have a hematopoietic organ. It was assumed that embryonic [62] and larval hemocytes are passed on and constitute the adult hemocyte population where they stop proliferating (reviewed in [75]). However, it has recently been shown that four hematopoietic clusters exist in the abdomen of the fly adjacent to the heart. The hemocytes are embedded within a 6 network of extracellular matrix where they develop from precursors to either plasmatocytes or crystal cells. Lineage tracing revealed that hemocytes of the secondary lymph gland lobes give rise to the precursors in the hematopoietic clusters. When the fly is challenged by bacterial infections, hemocytes divide in the hematopoietic hub. The authors stress the similarity with human bone marrow and suggest using the adult fly´s hematopoietic hub as a model system for human hematopoiesis [59]. Immune response to parasitoid wasps 1. 1. wasps inject eggs + venom 3. 2. 5. 4. 2. layer of on unknown compound is deposited on the egg 3. plasmatocytes are recruited and spread on the egg 4. lamellocytes are recruited to the egg and encapsulation ensues unknown compound plasmatocytes lamellocytes melanin deposit 5. melanin is deposited on the egg to seal it off Figure 3 The melanotic encapsulation response against wasp eggs. Long since have scientist been fascinated by the interaction of parasites with their hosts. Like many other insects, Drosophila is frequently infected by parasitoid wasps. Wasps from at least five genera parasitize Drosophilids. The figitids Leptopilina spp. and Ganaspis spp. as well as the braconids Asobara spp are cosmopolitan larval parasitoids, while Trichopria spp. (Diapriidae) and Pachycrepoideus vindemmiae (Pteromalidae) attack the pupal stages of fruit flies [89], [90]. In particular representatives of the genera Leptopilina, Ganaspis, and Asobara have been studied in the context of immunology. Melanotic encapsulation is evolutionary conserved in insects and is the visible evidence of the vigorous immune response that the host mounts in response to parasite infection. It is a stepwise process that comprises recognition of non-self surfaces, recruitment of blood cells, transcriptional, translational, and biochemical changes in blood cells and fat bodies, as well as alterations in the metabolism and physiology of host larvae (reviewed in [91], [25], and Figure 3). The female wasp pierces the cuticle of the host with her ovipositor and injects an egg along with venom into the hemocoel of the Drosophila larva. The venom often contains virus or viruslike particles and additional compounds that attempt to attenuate the immune response of the host [92]. In the larva, the parasitoid egg is recognized by an unknown mechanism. The first visible event in capsule formation is the deposition of an electron-dense layer of unknown material onto the chorion of the wasp egg as early as six hours after infection that is recognized by plasmatocytes [93]. This layer might consist of extracellular matrix because plasmatocytes of larvae mutated in LaminA were not able to adhere to wasp eggs [94]. Plasmatocytes are recruited from the sessile compartment [70], [63] and the lymph glands [31], [44]. They adhere to the electron dense layer, spread over the egg surface, and form septate junctions to seal of the egg [93]. Plasmatocyte recruitment after infection by Ganaspis sp.1 is dependent on a calcium burst that is controlled by a calcium channel encoded by 7 Rya-r44F (Ryanodine receptor 44F) [95]. Secretion of the cytokine edin (elevated during infection) from the fat body activates plasmatocytes to leave the sessile hemocyte compartment [96]. Spreading of plasmatocytes on the wasp egg and formation of septate junctions between plasmatocytes is mediated by the RhoGTPase Rac2 [97]. Twenty-four hours after infection, lamellocytes appear in circulation and start to encapsulate the egg [93]. RhoGTPases, RhoGEFs, and integrins are needed for proper lamellocyte function [98], [94] . Adhesion of lamellocytes to the plasmatocyte-covered wasp egg is controlled by the cellular adhesion molecule Neuroglian [99]. The RhoGEF Zizimin-related (Zir) mediates spreading of lamellocytes via the RhoGTPases Rac2 and Cdc42 [97], [100]. Rac1 in combination with the heat shock protein Hsp83 mediates the relocalization of the beta-Integrin Mysopheroid (Mys) to the periphery of spreading lamellocytes [101]. In addition, Rac1 signals via the Jun Kinase basket (bsk) to regulate the turnover of focal adhesions [102]. N-glycosylation on membrane proteins of lamellocytes is needed to consolidate the capsule [103]. In addition, Hemese (He), a membrane glycoprotein that is predicted to be O-glycosylated, is a negative regulator of encapsulation [104]. Finally, melanization of the encapsulated egg ensues, and the egg is completely entombed within several layers of lamellocytes and encrusted by melanin deposits forty hours after infection [105],[93],[97]. Rac1 and Rac2 mutant hemocytes, as well as RNAi of bsk in blood cells display defects in melanizing wasp eggs [97], [102]. Hemocytes are the key players in the encapsulation response. Many studies found that high larval blood cell numbers increased the success in killing the parasite [29], [106],[107],[108],[109]. Only one study that used 24 european field lines of D. melanogaster was not able to find that resistance to A. tabida was correlated with a high hemocyte load [110]. The importance of hemocytes for the encapsulation reaction becomes evident as D. obscura as is not able to generate lamellocytes in response to parasitization [111]. Drosophila as a research tool Next to the commonly advertised advantages of ideal model organism like comparatively low maintenance costs due to small size, simple diet, short life cycle, and fast reproduction, the true power of the fruit fly lies in its long and successful history in research and in a fly community eager to develop new tools and make new discoveries. It started in 1910, when T. H. Morgan introduced Drosophila into his laboratory to study heredity. Only within five years, Morgan and his students Alfred Sturtevant, Calvin Bridges, and Hermann Muller developed the theory of heredity (reviewed in [112]), [113]. During these five years, more discoveries were made that greatly contributed to the understanding of the principles of genetics for which Morgan was awarded the 1933 Nobel Prize in Physiology or Medicine. From a lab worker´s perspective, the introduction of balancer chromosomes by Muller in 1918 is one of the highlights (reviewed in [112]). Muller´s research on mutagenesis earned him the Nobel Prize in Physiology or Medicine “for the discovery of the production of mutations by means of X-ray irradiation" . Christiane Nüsslein-Volhard and Eric Wieschaus systematically mutated the Drosophila genome and screened for genes impacting early development of the fly [114], which won them the Nobel Prize in 1995 together with Edward Lewis "for their discoveries concerning the genetic control of early embryonic development". Traditional mutagenesis only permitted forward genetics. Transgenesis by transposable elements opened the door to reverse genetics. Allan Spradling and Gerry Rubin used the P-element as a “gene ferry” to artificially introduce extraneous DNA into the fly´s genome [115], [116], [115]. P-element-mediated transgenesis fueled the further development of additional techniques for gene manipulation. P-elements and piggyBacs are the most widely used transposons [117]. P-element or piggyBack insertions are available for more than 65 % of Drosophila genes. These transposons 8 can be directly used for gene disruption or to introduce targeted expression systems into the genome [117], [118], [119]. The FRT/FLP system adapted from yeast is based on the recombinase Flippase (FLP) to act on the Flippase Recombination Target (FRT). FRT/FLP is used to create deletions [120] and to induce mitotic homozygous clones in otherwise heterozygous animals [121], [122]. The GAL4-UAS system for targeted gene expression uses the yeast transcription factor GAL4 to bind to the promoter sequence UAS (Upstream Activation Sequence). GAL4 expression is regulated by tissue-specific promoters that control UAS-mediated gene expression [123]. GAL80, a negative regulator of GAL4, permits the tempo-spatial control of gene expression by the GAL4-UAS system [124], [125], [126]. MARCM (Mosaic Analysis with a Repressible Cell Marker) synergistically combines FLP/FRT, GAL4UAS, and GAL80 to mark homozygous clones [127], [128]. The Phi-C31 integrase is facilitates target-specific genome engineering [129]. Finally, the CRISPR/CAS technology has further eased genome engineering and introduced gene editing [130], [131]. Article III of this thesis is based on the development of a new generation of deficiency kits and the development of in vivo RNAi technology. Deficiencies are delations that span a few to some hundred genes. They can be maintained in Drosophila over balancer chromosomes. Deficiencies are Null alleles of the genes they cover. They have been used to map mutations and to carry out modifier screens [132]. Through the years, the Bloomington stock center has assembled a core collection of 270 deletion strains spanning 70 % of the genome. The disadvantages of these deletions are their heterogenous genetic backgrounds due to their diverse origins and their imprecise mapping to the genome sequence [133]. The DrosDel [134], [135] and Exelixis consortia [136] used genome engineering based on targeted recombination mediated by the FRT/FLP system carried in P element [120], [137] and piggyBac vectors [138] to overcome these limitations. The new deficiency kits were generated in isogenized genetic backgrounds and the breakpoints are precisely mapped to genome sequence [133]. The DrosDel deficiencies cover 77 % and the Exelixis collection 56 % of the genome. The Bloomington stock center created and additional set of deficiencies based on the Exelixis collection. In combination, the DrosDel, Exelixis, and the Bloomington stock center deficiencies cover 98.4 % of the genome [139]. RNA interference (RNAi) by double-stranded RNA is an efficient method of gene silencing [140]. It is widely used in many species for in vitro and in vivo RNAi. In Drosophila, in vivo RNAi is delivered by UAS-hairpin constructs that provide the siRNAs necessary for gene silencing. GAL4-directed expression of the UAS-hairpin constructs allows tempo-spatial control of RNAi. Drawbacks of RNAi are false positive and false negative results. Off-target effects that lead to false positives are caused by siRNAs that recognize and destroy mRNA of genes with similar nucleotide sequence, and weak GAL4 driver expression insufficiently knocks down genes what may result in false negatives [141], [142]. Three independent RNAi libraries approach the problem of off-target effects in different ways. Currently approximately 30000 UAS-hairpin constructs are available [141]. Genetic Screens in Drosophila with focus on hemocytes The purpose of a genetic screen is to identify new genes and their functions in a particular process. Screening strategies follow two approaches: forward and reverse genetics. In forward genetic screens, the wild-type function of a gene is inferred from its mutant phenotype. Mutations can be induced by radiation, chemical mutagens, or genetic techniques [132]. The genotypes of mutations fall into two classes: loss-offunction and gain-of-function mutations. Loss-of-function mutations are either amorphs with complete loss of gene function (also called Null mutations), or hypomorphs with reduced gene function. Gain-of-function mutations are 9 hypermorphs with enhanced gene function, neomorphs with novel gene functions, and antimorphs with gene function opposite to the original function (also called dominant negative mutations). Genetic screens in Drosophila have a successful tradition. In 1995 Christiane Nüsslein-Volhard and Eric Wieschhaus received a Nobel Prize for “Genetic control of early structural development”. They investigated mutations of the embryonic development of cuticular structures and identified segment polarity, pair-rule, and gap genes by aforward genetic screening strategy [114]. Several types of screens have been developed during the years (reviewed in [132], [143]). I will describe screening strategies that have been used to find unknown genes in Drosophila immunity and hematopoiesis. The first forward genetic screen to discover immunity genes that respond to bacterial infection was carried out by Wu et al. [144]. In reverse genetic screening, known mutations of a genotype can be used to screen for alterations in a specific phenotype. In vivo RNAi is a tool predestined to carry out reverse genetic screens. In blood cell research, in vivo RNAi was used to identify genes that induce lamellocyte formation [45] or regulate lymph gland hematopoiesis [145], [146], [147]. A particularly useful approach to decipher unknown genes in a signaling pathway is a genetic modifier screen. The key element of a modifier screen is a sensitized genetic background in the signaling pathway or developmental process under investigation. In a sensitized genetic background, an allele of a gene is used that partially disrupts gene function, either enhancing (hypermorph) or reducing (hypomorph) it. Amorphic mutations in most genes are recessive therefore only one gene copy is needed to secure gene function. Under this condition, the sensitized genetic background becomes susceptible to the signaling activity of downstream genes, which may cause phenotypically detectable changes. A gene rendering the phenotype of a sensitized genetic background more severe is called enhancer, a gene rendering it more wild-type-like is called suppressor. This approach was used to find genes involved in lymph gland hematopoiesis. A Zfrp8-Null allele with a lymph gland overgrowth phenotype was used as the sensitized genetic background and deficiencies were used to screen for enhancers and suppressors [146]. A misexpression screen is based on the assumption that also ectopic activation of genes can help to elucidate gene function. Transposons have been used to introduce promoter or enhancer elements into the fly genome that induce gene expression at the integration site. Pernille Rørth created the first misexpression library called EP (Enhancer Promoter) [148], additional libraries were created later [118]. Pxn-GAL4 induced overexpression of such enhancer elements was used to screen for genes involved in peripheral larval blood cell hematopoiesis and localization [85]. Methods for hemocyte research Originally, hemocyte types have been characterized by morphology and ultrastructure with the help of light and electron microscopy. Research on Drosophila blood cells started in larvae, presumably that is the developmental stage where hemocytes are most easily accessible. Plasmatocytes and crystal cells are the basic hemocyte types in all developmental stages of Drosophila [59], [149]. They are round cells with a diameter of 7 to 12 μm. Plasmatocytes have a smooth cytoplasm with primary lysosomes, and crystal cells harbor crystalline inclusions in their cytoplasm. In addition, smaller cells with a diameter of 4 to 8 μm are referred to as prohemocytes. Lamellocytes are large flat cells with a diameter of up to 50 μm but with a thickness of only 1 μm. Their cytoplasm contains only few organelles, and their plasma membrane often has microridges [30], [32], [31]. Advancements in molecular biological and genetical techniques enabled the systematic identification of molecular markers for hemocytes. Screening of enhancer trap libraries for β-galactosidase expression in circulating and lymph gland 10 hemocytes in the embryo and larva identified some of the first genes expressed in blood cells. However, hemocytes were often not the only β-galactosidase-expressing tissue present in individual fly lines [150], [151]. Kimbrell and coworkers screened for reporter expression in immune tissues and for reporter lines with increased expression induced by bacterial infection. They found Dorothy (Dot), escargot (esg), and Cytochrome b5 (Cyt-b5) to be expressed in lymph glands as well as Collagen 25C (Cg25C) and viking (vkg) in hemocytes and fat body. Esg and Cyt-b5 lines had melanotic tumors. Thor expression increased after bacterial challenge [150]. Meister and coworkers looked for enhancer trap lines with activity predominantly in lamellocytes. They discovered 21 lines with hemocyte expression patterns, and five of these could be used as lamellocyte reporters. The lamellocyte-specific reporter lines were mapped to misshapen (msn), cAMP-dependent protein kinase A (Pka-C1), Chip (Chi), and CG9932. An addition, they found domino [151]. The insertion close to msn has been used as a lamellocyte reporter. Another approach was to generate monoclonal antibodies against hemocyte antigens that were used to identify hemocyte-specific genes. PDGF- and VEGFrelated factor 2 (Pvf2) was discovered by purifying the antigen with the help of affinity chromatography and protein sequencing [77]. The Ando and Hultmark labs generated pan-hemocyte antibodies, and antibodies specific for plasmatocytes and plasmatocyte subtypes, lamellocytes and lamellocyte subtypes, as well as crystal cells [72]. They identified five genes by using these antibodies to screen cDNA expression libraries [104]. Hemese (He) is expressed by all hemocyte types in the periphery and in the lymph glands. It is predicted to be glycosylated and has sequence similarity with Glycophorins on the cell membranes of erythrocytes. He is a negative regulator of the encapsulation response to parasitoid wasps and of tumor severity of l(3)mbn [104]. NimC1 is expressed on all plasmatocytes. It is a phagocytosis receptor and the founding member of a cluster of ten structurally related nimrod-like genes on chromosome arm 2L [152]. Atilla is expressed by all lamellocytes. Its expression is upregulated twofold in Ras-induced overproliferating hemocytes [153]. Cheerio (cher) codes for Filamin-240, which is involved in the reorganization of the actin cytoskeleton during the flattening of lamellocytes [154]. The L4 antigene was identified as a beta-PS integrin (Kurucz et al. unpublished in [75]). In the fly community, the He/H2, NimC1/P1, and atilla/L1 antibodies have become the defining tool for blood cells in general, as well as plasmatocytes and lamellocytes in particular. The characterization of molecular markers for hemocytes permitted the construction of hemocyte-specific GAL4 driver lines. He-GAL4 was the first driver expressed in all larval hemocyte types, but only in approximately 80 % of all circulating hemocytes. In contrast to its antigen, it is not active in the lymph glands of unchallenged larvae [70]. Hemolectin-GAL4 (Hml-GAL4) [69], its truncated version HmlΔ-GAL4 [155], Peroxidasin-GAL4 (Pxn-GAL4)[156], eater-GAL4 [157], srpHemoGAL4 [158], and Croquemort-GAL4 (crq-GAL4)[159] are expressed in embryonic plasmatocytes, in larval plasmatocytes and crystal cells in circulation and in the lymph glands, whereas eater is not to to expressed in the embryo [160]. Cg-GAL4 is expressed in plasmatocytes and the fat body [153]. Lz-GAL4 is specific for crystal cells [161], [43]. He-GAL4, Hml-GAL4, Pxn-GAL4, and crq-GAL4 are also expressed in the hematopoietic hub of the adult fly [59]. Hml has sequence homologies with the von Willebrand factor and is involved in hemolymph clotting [69]. Pxn [162] and Cg25C [40] are extracellular matrix molecules produced by hemocytes and the fat body. Crq and eater are phagocytosis receptors of which Crq mediates the phagocytosis of apoptotic bodies [163] and eater of bacteria [160]. Srp [164] and lz [161] are transcription factors involved in hematopoiesis. Besides ectopic expression of transgenes, these GAL4 drivers enable the targeted expression of fluorescent proteins 11 in hemocytes to study expression patterns of hemocyte compartments and to monitor hemocyte behavior in vivo. In addition to GAL4 drivers, hemocyte-specific enhancer-reporter transgenes were generated that decouple hemocyte visualization from driver expression. These constructs facilitate the investigation of signals from other immune tissues in hematopoiesis and immunity. Fly lines are available for each blood cell type and tagged with different fluorescent proteins. This makes it possible to visualize several blood cell types in combination [157]. The eater [165] and Hml [64] enhancers were used to construct plasmatocyte-specific reporter constructs. The MSNF9mo enhancer within msn [166] and the Bcf6 enhancer upstream of PPO1 [167] direct expression specifically to lamellocytes and crystal cells, respectively. An insertion of a Minos element into the atilla gene can be used as a lamellocyte reporter [168]. Recently, Ando and coworkers combined in vivo antibody staining with blood cell reporter expression for live imaging of hemocytes in Drosophila larvae [169]. Many more tools are available for the lymph glands and are summarized in a current review [170]. Circulating larval hemocytes have generally been counted with the help of hemocytometers (for example [70]) which is tedious, time consuming, and errorprone. A flow cytometry-based approach for circulating and lymph gland blood cells was introduced [171], but only one report so far used flow cytometry for quantifying blood cells [43]. In the adult fly, FACS was applied to sort GFP-expressing blood cells [172], [38]. Moreover, an antibody-based rosetting method to separate hemocyte subpopulations was developed [173]. 12 Aims of the thesis The general aim of this thesis has been to find out how the immune system controls and orchestrates the hematopoiesis of lamellocytes. Lamellocytes constitute a unique cell type in the respect that they are not present in the blood cell repertoire of healthy larvae. Instead, their development is induced “naturally” - by wasp infection, or “artificially” - under aberrant genetic conditions that lead to melanotic nodules also referred to as tumors. Anyhow, their appearance signals that the individual is immune-compromised. I used genetics, imaging, and flow cytometry to find out more about lamellocyte hematopoiesis. In Article I we used the blood cell driver He-GAL4 in a directed overexpression screen to investigate which signaling pathways are involved in the activation of the cellular immune system. Specifically, we looked for changes in numbers of peripheral plasmatocytes and crystal cells and for the induction of lamellocytes. In Article II we aimed to develop methodology to automate hemocyte counting and facilitate live imaging of hemocytes in Drosophila larvae. We focused on a reporter construct-based approach that enabled counting of hemocytes and distinguishing between hemocyte subtypes by flow cytometry and live imaging. In Article III we developed a genetic modifier screen to identify new genes that control the activation of the cellular immune system. In Article IV we studied the mechanism of Toll-dependent activation of blood cells. 13 Article I “A directed screen for genes involved in Drosophila blood cell activation.” Preface In parallel to their work on humoral immunity, the Hultmark lab was aiming to create antibodies against hemocytes to study cellular immunity. Istvan Ando and Eva Kurucz had already generated some monoclonal antibodies and joined the Hultmark lab, where a larger set was generated and characterized. This collaboration led to the discovery of Hemese [104]. Since the Hemese antibody stains all hemocyte types [72] it was decided to generate a Hemese-GAL4 driver assuming it would be expressed in all hemocytes. I joined the lab at the end of 2002 when Calle Zettervall had already cloned HeGAL4 and had started to overexpress genes relevant for human cancers. My contribution to Article I was setting up a method for differential hemocyte counting and then counting larval hemocytes of the F1 generation of He-GAL4-induced ectopic expression of the transgenes that induced visible immune phenotype. Michael Williams counted sessile crystal cells. Summary of main results 1. The Hemese-GAL4 driver can be used to ectopically express genes in blood cells. 2. Ectopic expression of activated and wild-type constructs of different signaling pathways induce fate changes of hemocytes and affect proliferation of plasmatocytes and activation of lamellocytes. Discussion 1. The He gene was identified by using monoclonal antibodies previously generated against larval hemocyte to screen a cDNA expression library made from l(3)mbn-1 larval blood cells [104]. The Hemese antibody is specific for all sessile and circulating hemocyte types as well as for hemocytes residing in the lymph glands [72]. The He-GAL4 driver was the first pan-hemocyte driver generated. Its expression starts during the second larval instar (IA unpublished observation). The He-GAL4 driver is active in all blood cell types, but only in 75-80 % of all circulating blood cells. It is also expressed in sessile hemocytes. As circulating and sessile hemocytes seem to be one interchangeable population [64], it is likely that the percentage of He-GAL4expressing cells is equal in both compartments. In contrast to the antibody, He-GAL4 is not expressed in lymph glands of non-immune challenged larvae. We used HeGAL4 to overexpress genes that have been identified in human cancers. 2. We have started to use the terminology proliferation for increase of plasmatocytes and activation for the de novo generation of lamellocytes. Several models for lamellocyte activation have been proposed (see introduction and discussion of Article II). According to our model in Article II, two lineages exist in peripheral hemocytes: the plasmatocyte and lamellocyte lineage. Plasmatocytes are extraordinarily plastic. After wasp parasitization three types exists. Lamellocytes are generated in a demand-adapted manner from what we assume are prohemocytes. Prohemocytes that undergo avid proliferation develop via prelamellocytes to mature lamellocytes. Increase in total hemocyte count during the beginning of the encapsulation response is almost entirely due to the proliferation of the lamellocyte lineage (see discussion of Article II). Expression of a wild-type form of Alk, an active 14 form of Pvr, and the Tl10b mutant induce similar phenotypes when monitored with the double hemocyte reporter construct eaterGFP,msnCherry (IA unpublished results). As the blood cell phenotypes are similar after parasitization and genetic manipulation, the results obtained in Article III can be used to reinterpret the blood cell counts in Article I. Forced expression of constructs that represent different signaling pathways turn on either a proliferation program, an activation program, or a combination of both. When plasmatocytes strongly proliferate, normally lamellocyte activation does not occur like in the case of Egfr, Ras85D. Plasmatocyte diameters after overexpression of Egfr and Ras85D visibly decreased. Small plasmatocytes were also observed when Cg-GAL4 was used to express Ras85D.V12 [153]. The small size of these cells is compatible with a hypothesis that these cells are prohemocytes and constitutive Egfr-Ras85D signaling maintains prohemocyte fate and is necessary for prohemocyte proliferation. Also adult midgut progenitor cells require Egfr signaling for proliferation [174]. If the immune response favors activation as after the expression of an activated form of hemipterous (hep.CA), the Drosophila homolog of Jun kinase kinase (JunKK), or hopscotch (hopTum), the Drosophila homolog of Jak, only the lamellocyte lineage is activated. Jak/Stat signaling is activated during the encapsulation response [175], [176], [177]. Negative regulation of the wingless pathway also mainly activates lamellocytes without a strong proliferative response of plasmatocytes. This is in line with the role of wingless signaling in stem cell maintenance in the lymph gland [178]. The expression of a gain-of-function construct of shaggy (sggS9E), the Drosophila homolog of Gsk3, had a particularly interesting overgrowth phenotype of the secondary lymph gland lobes. Overexpession of Alk, Pvr, and Toll activates the lamellocyte lineage and induces proliferation of plasmatocytes. However, in comparison to Egfr and Ras85D expression, plasmatocytes are diverse. High numbers of activated plasmatocytes type II (see Article III) are in circulation. These cells are very reactive. And it is therefore possible that the high degree of larval and pupal lethality is due to the presence of this cell type. Notably, several signaling pathways induce similar phenotypes. This suggests that signaling pathways either act in parallel or sequentially. Sequential activation can occur within the same cell or tissue or between cells or tissues. Cytokines are needed for tissue interaction, which I discuss under Article IV. As overexpression of the Junk and the Jak/Stat signaling pathway components had similar phenotypes, we tested if these two pathways interacted genetically. We found that the Jak/Stat pathway signals downstream of the Junk pathway to activate lamellocytes, and that the Jak/Stat pathway suppresses all the effects of activated Junk signaling such as lamellocyte activation, loss of sessile cells, and melanotic nodules (Kronhamn et al.unpublished manuscript). This is supported by a report of the Schulz lab [166]. Moreover, serpent (srp) activity promotes lamellocyte activation downstream of the activated Jak/Stat pathway, whereas u-shaped (ush) suppresses lamellocyte fate [76]. Some signaling pathway interactions are known for the humoral immune response [179], [180], [181]. 15 Article II “Infection-induced proliferation is a central hallmark of the activation of the cellular immune response in Drosophila larvae.” Preface and study design The quantification of hemocytes for Article I in the traditional manner by hemocytometer led to the insights that blood cell numbers are variable, thus many individual larvae need to be counted to obtain reliable results. Moreover, cell counting in that way is not an entirely objective method, because it depends to a great extent on the skill and patience of the experimenter. To circumvent these obvious flaws, in Article II, we combined fluorescent plasmatocyte- and lamellocyte-specfic enhancer-reporter constructs and developed a flow cytometry-based approach for cell counting that we verified by confocal microscopy and cell sorting. In addition, these reporters facilitated live imaging of Drosophila larvae. Fluorescent hemocyte type-specific enhancer-reporter constructs decouple GAL4UAS driven gene expression from the mere observation of hemocytes. We had started to design our own enhancer-reporter constructs just prior to the Schulz lab´s report of fluorescently tagged plasmatocyte, crystal cell, and lamellocyte constructs [157]. We abandoned our own approach and instead used their constructs. I meiotically recombined plasmatocyte and lamellocyte reporters tagged with different fluorescent proteins onto the same chromosome. I also generated fly lines of plasmatocytelamellocyte reporter constructs in combination with several independent tissuespecific drivers and fluorescent constructs reporting other cell functions. In all experiments, we were careful to have only one copy of each enhancerreporter, UAS-, and GAL4 construct in the F1 generation to warrant identical genetic background compositions. This was necessary because differing genetic backgrounds add another layer of complication to the already variable blood cell counts and influence the speed of the immune reaction. For example in Fig. 9 of Article II, the appearance of eaterGFP-low plasmatocytes in the RNAi-mediated knockdown of edin in the fat body was delayed by three to four hours in a homozygous enhancer-reporter genetic background, compared to the heterozygous genetic background. Lamellocyte activation is part of the immune response induced after parasitization by parasitoid wasps. The study of the virulence factors wasp females inject alongside the egg into fly larvae bring to light the importance of the encapsulation reaction for fly survival, as virulence factors attempt to abrogate this immune response to ensure the survival of wasp larvae (reviewed in [25]). Because we wanted to study lamellocyte hematopoiesis with a combined approach of flow cytometry and imaging, we deliberately chose three wasp species of the genus Leptopilina that affect the cellular immune response in different ways. L. boulardi is a specialist for D. melanogaster and kills approximately half of the infected individuals. L. heterotoma is a generalist species that kills almost all D. melanogaster [177] by lysing lamellocytes [182], [183]. D. melanogaster is not a host-species for L. clavipes and hence kills L. clavipes [184]. We expected that the predispositions of these wasp species would be reflected in the dynamics and the cell types participating in the induced immune response in fly larvae. Many features of the encapsulation response have been described (reviewed in [25]), however, there has never been a study that takes a holistic view of the dynamics of blood cells at the different sites in the larva. We therefore decided to investigate circulating and sessile blood cells, the blood cell response on the wasp egg, and the stability of the lymph glands at different time points in non-infected and infected Drosophila larvae. The time points for the survey of sessile blood cells and for the blood cell response on the wasp egg were determined by time points of the timeline of circulating cells that mark major 16 changes. I constructed all the genetic tools used in this project. Laura Vesala and I conducted all experiments together. Summary of main results 1. Flow cytometry with two different reporter constructs detects six different blood cell types of two functionally different lineages and a negative cell population: Plasmatocyte lineage: eaterGFP-high plasmatocytes, activated plasmatocytes type I and type II Lamellocyte lineage: eaterGFP-low plasmatocytes, prelamellocytes, and lamellocytes 2. Wasp parasitization induced a demand-adapted hematopoiesis in circulating hemocytes that was based nearly only on the proliferation of eaterGFP-low plasmatocytes and prelamellocytes of the lamellocyte lineage. 3. The origin of eaterGFP-low plasmatocytes remains elusive. 4. The encapsulation response against all wasp species is identical. It seems to be the virulence factors of the wasp species rather than the immune response of the fly that determine the outcome of infection. 5. The concerted action of hemocytes of all compartments (circulating and sessile hemocytes, hemocytes on the wasp egg) contributes to a successful immune reaction. 6. The depletion of the cytokine edin in the fat body reduced eaterGFP-low plasmatocyte numbers. Discussion 1. Flow cytometry has a long history in human blood cell research [185]. The development of flow cytometers that can distinguish between 12 different fluorophores plus forward and side scatter was instrumental for modern blood cell research and diagnostics. More than 100 blood cell populations can be identified from human blood [186]. In Drosophila, only one report attempted to characterize different blood cell populations simultaneously [171]. In this report, the distinction between different blood cell types relied to a great extent on immunohistochemical methods. Considering the fact that individual larvae have a low volume of hemolymph, immunohistochemical methods for blood cell type differentiation and counting are not suitable for high-throughput analysis on the level of individual larvae. We are the first laboratory to develop a flow cytometry technique based on two fluorescent in vivo reporter constructs [166], [165]. This enabled the detection and counting of six different blood cell populations. In addition, we used flow cytometry for detection and counting of proliferating cell types with an experimental set-up of three fluorescent proteins. Key to using flow cytometry is not only the development of adequate genetic and other tools to investigate cell functions, but also the appropriate flow cytometer. We were lucky to have an AccuriC6 flow cytometer at the institute. The advantage of the AccuriC6 is the low volume of fluid it requires. We routinely prepared 100 µl of a blood cell suspension of which we analysed 30 µl. This small amount of fluid was enough to obtain reliable results. Flow cytometry-based counting of distinct hemocyte populations can still be improved. The AccuriC6 is also available with three lasers, which would allow the detection and separation of additional fluorescent reporters. It would be desirable to better characterize the hemocyte subtypes and create additional fluorescent in vivo enhancer constructs. 2. In accordance with our findings, I suggest to distinguish between steady-state hematopoiesis in healthy and demand-adapted hematopoiesis in immune-challenged animals because these processes need to meet different requirements and are functionally different. Steady-state hematopoiesis provides the animal with blood 17 cells for the daily wear-and-tear, whereas under circumstances of demand, new effector cells have to be created and cells need to proliferate quickly (reviewed in [187]). Immune responses require extra energy [188]. In addition, effector cells are potentially harmful to the animal itself. Thus restricting the hematopoiesis of effector cells such as lamellocytes contributes to immune and energy homeostasis. In this study, we show that plasmatocytes develop by steady-state hematopoiesis regardless of the state of the immune system, and lamellocytes by demand-adapted hematopoiesis. However, after wasp infection eaterGFP-high plasmatocytes displayed an extraordinary plasticity. In unchallenged larvae, eaterGFP-high plasmatocytes were the predominant plasmatocyte type, thus the plasmatocyte population appeared very homogenous. Plasmatocyte counts increased steadily during the time course of our experiment and at the end of the time line, a sudden increase presumably due to changes in the hormonal status just before metamorphosis occurred [189]. We were not able to follow crystal cell counts with our experimental set up. After infect by parasitoid wasps, plasmatocytes display a remarkable plasticity in circulation and on the wasp egg. Activated plasmatocytes type II developed in contact with the wasp egg and activated plasmatocytes type I in circulation and within the sessile islets. Activated plasmatocytes type II retain eaterGFP and NimC1 expression while also requiring lamellocytes markers. They seem to represent a new cell type that unifies plasmatocyte and lamellocyte features. Lamellocytes are immune-effector cells that only develop under certain conditions. The mode and place of their hematopoiesis remains controversial. Rizki described lamellocytes as morphological variants of circulating plasmatocytes that emerge at the end of the third larval instar [30]. This view was corroborated by three independent lineage tracing experiments that show that lamellocytes develop from plasmatocytes [45], [44], [46] and one publication reporting that lamellocytes develop from sessile plasmatocytes [63]. In contrast, other reports claim that the lymph glands are the only source of lamellocytes. In the lymph glands, lamellocytes develop from prohemocytes controlled by the actions of cells in the posterior signaling center. The lymph glands disintegrate and the lamellocyts are released into the hemolymph [81]. Yet another publication assumes that lamellocytes can develop from prohemocytes also in circulation, but they are mainly released from the lymph glands [31]. In our study, we show that lamellocytes develop in a demand-adapted fashion. Early after infection eaterGFP-low plasmatocytes appear in circulation and divide avidly. They develop further into prelamellocytes that give rise to mature lamellocytes. Because of their sudden appearance, the low and transient expression of eaterGFP, and the high proliferation rate, we assume that these cells are prohemocytes or derive from prohemocyte. 3. The appearance of eaterGFP-low plasmatocytes is our most interesting discovery. We tried hard to unravel their origin, but were not able to solve that quest. As described before, two experimentally based hypotheses of the origin of lamellocytes have been discussed in literature. Therefore we considered several possibilities for the origin of eaterGFP-low plasmatocytes Firstly, eaterGFP-high plasmatocytes give rise to eaterGFP-low plasmatocytes by asymmetric cell division, by dilution of the eaterGFP content due to an accelerated cell cycle accompanied by a strong increase in proliferation rate, or by simply down regulating their eaterGFP while directly transdifferentiating into eaterGFP-low plasmatocytes. We tested the first option by imaging blood cells of infected larvae early during the immune response in order to find cells with high and low eaterGFP expression that had just undergone mitosis. We saw many dividing cells, but none met the requirement of asymmetric cell division. Next, we considered the dilution effect of eaterGFP after cell division. The fluorescent signal emitted by eaterGFP-high plasmatocytes is approximately ten times higher than that of eaterGFP-low plasmatocytes. This 18 implies that eaterGFP-high plasmatocytes have to go through three to four rounds of cell division within only eight hours (the time of the first appearance of eaterGFP-low plasmatocytes until their highest peak). This also implies that eaterGFP-high plasmatocytes must quadruplicate their division rate to keep their own numbers constant while serving as a pool for eaterGFP-low plasmatocytes. Alternatively, the counts of eaterGFP-high plasmatocytes should drop drastically in favor of eaterGFPlow plasmatocytes during the early demand-adapted phase of the immune reaction. None of the listed options is in agreement with our experimental data. Finally, the half-life of enhanced GFP is approximately 24 hours [190], [191]. Down regulating enhanced GFP in only eight hours is impossible. Secondly, eaterGFP-low plasmatocytes are prohemocytes or derive from prohemocytes only transiently expressing low levels of eaterGFP during their maturation step to become lamellocytes. The most compelling support for this hypothesis is that plasmatocyte and lamellocyte lineages are strictly separated on our FACS plots. Moreover, several publications report the presence of prohemocytes [31], [192], [193] in circulation. 4. The interaction of a parasite with its host has always been described as an evolutionary arms race. Some of the virulence mechanisms of the wasp parasitoids have been described and the encapsulation response has been studied (reviewed in [25]). When comparing the differential hemograms of circulating hemocytes of animals infected by the three different wasp species, it becomes evident that the encapsulation response is orchestrated in a similar manner with slight delays in the timing. It rather seems that the virulence factors of the wasps shape the immune response. In the lamellocyte lineage after a lapse phase with no apparent action, eaterGFP-low plasmatocytes, prelamellocytes, and lamellocytes appear stepwise in circulation. After all these cell types have reached their peaks, their numbers drop and even out at a lower level. The timing of eaterGFP-low plasmatocyte emergence in the hemolymph and the appearance of the first blood cells on the wasp eggs of L. boulardi coincide. Blood cells do not adhere to eggs of L. clavipes and L. heterotoma. As aforementioned, L. heterotoma kills as good as all infected Drosophila larvae. L. heterotoma females inject lamellolysin alongside the egg into the host. Lamellolysin is a compound that affects lamellocyte morphology and finally kills them [194]. In our experiment, lamellocyte precursors, the eaterGFP-low cells, appear but the more mature lamellocyte types are depleted after infection with L. heterotoma. L. boulardi injects three venoms with know function into the host larva: A RhoGAP [195], [196] which affects hemocyte spreading behavior, a serpin [197], and a superoxide dismutase [198] both of which affect the melanization reaction. Larvae infected by L. boulardi have a clear delay in melanization compared to infection by L. clavipes. After L. clavipes infection, melanization of the wasp egg starts as soon as lamellocytes appear. 5. Simultaneously with the first eaterGFP-low plasmatocytes in circulation, eaterGFP-high and activated plasmatocytes type II are found on the wasp egg. This may suggest that recognition of the wasp egg by eaterGFP-high plasmatocytes induces the demand adapted-hematopoiesis of lamellocytes. In the wound healing response of the embryo, macrophages in the vicinity of the wound migrate towards wound edges [156] while in the wound healing response of third instar larvae hemocytes are recruited by direct capture from the hemolymph [20]. Pupal macrophages are able to crawl [199]. In embryos, H2O2 is generated at sites of wounding which induces a calcium flash in epithelial cells that directs macrophages to the wound (reviewed in [200]). Recently, Evans et al. published that the ITAM motif of Draper in combination with Src42A and Shark is required for recognition of H2O2 and migration of macrophages to wounds [201]. Currently it is not known in which manner plasmatocytes are recruited to the wasp egg. But while analyzing the images of wasp-infected larvae at early time points, I often had the impression that in 19 the vicinity of the wasp egg sessile hemocytes had vanished. This may suggests that plasmatocytes actively migrate towards the wasp egg from the surrounding sessile compartments. Additional experiments are needed to clarify how plasmatocytes are recruited to the wasp egg at the beginning of the encapsulation response. With live imaging of cultured wasp eggs, we were able to show that eaterGFP-high plasmatocytes differentiate into activated plasmatocytes type II on the wasp egg. Thus this differentiation process is tightly linked to the wasp egg. In addition, plasmatocytes type II are rarely seen in circulation and in the sessile compartment. The signals inducing these changes are so far unknown. Thirty hours after infection, the count of activated plasmatocytes type I rises over that of eaterGFP-high plasmatocytes. We are not certain, if these type of activated plasmatocytes phagocytizes msnCherry-containing material or if these cells truly express msnCherry. Their increase coincides with the hatching of the L. boulardi wasp larvae. Lamellocytes develop by demand-adapted hematopoiesis from prohemocytes. How the lamellocytes are recruited to the wasp egg is not known. However, we are able to show that blocking the secretion of the small peptide edin from the fat body reduces eaterGFP-low plasmatocytes. Therefore, the fat body has to receive a signal that instructs the secretion of edin. The nature of that signal is unknown, however, possibly dependent on Toll signaling in the fat body (see discussion in Article IV and [202], [203]). The initial signal how the fat body is alerted to the presence of the wasp egg is unknown. Also the lymph glands are alerted to the presence of the wasp egg. After infection of Drosophila larvae by L. boulardi and L. clavipes, the primary lymph gland lobes disintegrate. How the lymph glands sense the presence of a wasp egg is not known. A study by Sinenko et al. suggests that circulating plasmatocytes detect the wasp egg and signal to the lymph glands. In the medullary zone, the inhibition of Jak/Stat signaling in prohemocytes is released and ROS levels increase in the posterior signaling center. This has two effects: On the one hand, together with the inhibition of Jak/Stat signaling, ROS induces the differentiation of prohemocytes to lamellocytes in the medullary zone of the lymph gland. On the other hand, increased ROS levels induce the secretion of spitz, a ligand of Egfr, from the posterior signaling center into the hemocoel. Spitz then controls the transdifferen-tiation of plasmatocytes into lamellocytes [192], [204]. It is clear that a concerted action of all blood cell compartments, including those blood cells on the wasp egg, contribute to the encapsulation response. The origin and the nature of many of the signals are however still elusive. The development of new genetic tools is needed to unravel the interaction of different tissues. 20 Article III “Genetic screen in Drosophila larvae links ird1 function to Toll signaling in the fat body and hemocyte motility.” Preface As Article I of this thesis shows, several genes of distinct signaling pathways induce lamellocyte formation. In comparison to crystal cells and plasmatocytes that are present at all developmental stages, lamellocytes are only activated in immunocompromised animals in a demand-dependent manner. Lamellocytes are immune effector cells whose hematopoiesis is likely controlled differently from that of plasmatocytes and crystal cells. We have been interested in lamellocytes because of their role in encapsulation and melanotic nodule formation. A genetic screen seemed a reasonable approach to discover genes involved in lamellocyte hematopoiesis. My contribution to this project was designing the screen, developing the screening strategy, and constructing genetic tools. Martin Schmid and Jesper Kronhamn contributed to laying out some parts of the screening strategy. And Martin conducted the screen and did most of the work on ird1. I will therefore focus my discussion on the design of the screen and the screening strategy. Summary of main results 1. The Toll10b gain-of-function mutation can be used as a sensitized background for a genetic modifier screen. 2. Ird1 rescues the pattern of sessile bands and the loss of the primary lymph gland lobes, but enhances all other Toll10b phenotypes. Discussion 1. Genetic modifier screens have been successfully used to unravel genetic interactions and to discover new genes within signaling pathways (reviewed in [132]). The key to success is a sensitized genetic background that can be modified by mutations in genes that genetically interact with the gene providing the sensitized genetic background. In the work described in Article I, we show that the directed expression of a number of genes induced lamellocytes. Lamellocyte activation is usually accompanied by a change in the pattern of sessile hemocytes and melanotic nodule formation. Thus our criterion was that any gene whose overexpression induced melanotic nodule formation might provide a suitable sensitized genetic background for screening. I initially designed genetic tools for three sensitized genetic backgrounds based on UAS-Alk, UAS-Pvr.λ, and the Toll10b (Tl10b) mutation [205], [206]. All three genes have in common that they are receptors. Their overactivation induces gain-of-function genotypes, which are phenotypically characterized by the presence of lamellocytes, melanotic nodules, changes in the orderliness of the pattern of sessile hemocytes, and a mild to strong increase in plasmatocyte counts. Hierarchically, a receptor is the first gene in a signaling cascade within a cell, and mutations that decrease the functions of genes signaling downstream of the receptor are theoretically able to enhance or suppress the increased signaling activity of a genetically activated receptor. This is advantageous for screening because the yield of modifiers should be high. However, the outcome of the screen depends on the nature of the gain-of-function allele used (for discussion see [132]). Our screening strategy was to conduct an F1 deficiency screen with DrosDel deficiencies [134], [135], map the loci to single gene level with in vivo RNAi lines from 21 the VDRC (Vienna Drosophila Resource Center) [207], and confirm the newly discovered genes with mutations. Experiments we conducted earlier pointed out the importance of keeping the genetic background constant. This is due to the very nature of blood cells. Their numbers vary during larval development and between individuals of the same age and the same fly line. Because of this variability, a high number of individuals need to be screened to obtain interpretable results. To minimize the workload and make results more reliable that is to reduce the amount of false positives, keeping the genetic background constant and controlled is of paramount concern. The DrosDel deficiency kit was created in an isogenized genetic background, which tremendously helped in reducing the risks of false positives. The advantage of RNAi lines over mutations is that there exists at least one RNAi line for each gene in the fly. Moreover, the original GD lines of the VDRC RNAi library were generated in the genetic background of the DrosDel deficiency kit. In contrast, mutations come with diverse genetic backgrounds. The disadvantage of using RNAi is that deficiencies are functional null mutations and thus affect every tissue. Gene knockdown by RNAi is dependent on the GAL4-UAS system and therefore only affect those tissues the driver is active in. This implicates that whenever using RNAi lines to map loci determined on the basis of deficiencies, genes will be false positives, if their effects on the sensitized genetic background is dependent on a tissue the GAL4 driver is not expressed in. Originally, I had planned to screen for the modification of two phenotypes: the pattern of sessile cells in larvae and the melanotic nodule phenotype in larvae and imagines. In fact, screening for modification of melanotic nodules in imagines was my preferred strategy. To be able to visualize sessile blood cells in larvae and to be able to express RNAi lines in blood cells, I crossed HmlΔ-GAL4, UAS-GFP (HmlΔ>GFP) [155] into the fly line containing the Tl10b mutation. This generated w;HmlΔ>GFP;Tl10b/T1;3(OR60)/TM6,Tb. The Tl10b mutation is dominant female sterile [205]. Therefore, the Tl10b mutation is kept only in males to preserve the mutation and propagate it from generation to generation. For this reason, the Tl10b mutant fly line essentially consists of two individual fly stocks: w/Y; Tl10b/TM6,Tb (males) and w; T1;3(OR60)/TM6,Tb (females). TM6 is the balancer chromosome (Third Multiple 6) the Toll mutation is kept over. T1;3(OR60) is a translocation between chromosome one and three, which causes lethality in males. Crossing together these males and females produces only one viable type of male: w/Y; Tl10b/TM6,Tb and three viable types of females of which only one is fertile: w; T1;3(OR60)/TM6,Tb. In order to cross any construct into the Toll10b stock, it has to be done separately for each sex. HmlΔ-GAL4 (HmlΔ>GFP;Tl10b) drives GFP expression only in plasmatocytes without ectopic expression in salivary glands or the gut that hampered the use of the He driver for this screen. However, the viability of this newly created HmlΔ>GFP;Tl10b was lower than the original Tl10b stock. It was especially low when crossed to DrosDel deletions. I screened DrosDel deletions for the entire X-chromsome for enhancers or suppressors of nodule formation in the adult fly. It turned out that pupal lethality was too high. I was never able to recover more than ten or twenty flies per cross. This number of flies was insufficient to base a reliable screen on. Thus we abandoned this approach and turned to larvae, as larval viability was not as severely affected. DrosDel deficiencies are kept over balancer chromosomes with genetic markers that affect only structures visible in flies. Therefore, I crossed green balancer chromosomes (balancer chromosomes that express GFP also visible in larvae) of all chromosomes into the isogenized genetic background of the DrosDel kit. The GFP expression pattern of these green balancers differs from HmlΔ>GFP. All deficiencies were rebalanced over green balancers and used to distinguish deficiency chromosomes from balancer chromosomes in the F1 generation. In addition, most of 22 the fly stocks used in this project whose genetic backgrounds differed from that of DroDel were backcrossed six times into the genetic background of the DrosDel deficiency kit. Martin tested that mutations of MyD88, an adaptor protein within the Toll signaling cascade, suppressed the disruption of sessile cells, which verified our approach. Martin, Jesper, and I developed the hemocyte mobilization index based on a publication by the Li lab [208]. Because chromosome 3R had a high coverage rate with a relatively low number of individual deficiencies, we decided to screen this chromosome arm. In the end, Martin had screened 88 deficiencies, uncovered six suppressor regions, and discovered ird1. Ironically, mutations of ird1 also have an activated immune phenotype. However, the pattern of sessile cells and he loss of the primary lymph gland lobe is rescued. 23 Article IV “Control of Drosophila blood cell activation via Toll signaling in the fat body.” Preface In addition to designing and contributing to the Toll screen, I conducted a modifier screen of my own specifically tailored to discover novel genes involved in the activation of lamellocytes. Notably in this screen, deficiencies that uncover genes in the Toll signaling pathway were overrepresented. According to my strategy to use in vivo RNAi to map these loci to the single gene level, I knocked down genes of the Toll signaling cassette in hemocytes. However, RNAi-dependent depletion of Toll signaling in hemocytes did not suppress the phenotypes I was screening for, whereas mutants of the Toll signaling pathway were suppressors. Additionally, hemocytespecific expression of the RTKs Alk and Pvr, as well as other genes that induce lamellocyte activation, induced the activation of Toll signaling in the fat body measured by Drosomycin-GFP activity. Taken together, these findings suggested that there must be a signal generated in hemocytes that activates Toll signaling in the fat body, which would induce the expression of Toll-dependent genes that in turn would control hemocyte behavior. At the same time, Martin discovered in a joint project with the Rämet group that hemocyte-specific knock-down of MyD88 was not sufficient to rescue the melanotic nodule phenotype nor the aberrant pattern of sessile cells of the Tl10b mutation. Thus we joined forces to get to the bottom of the tissue specificity of Toll signaling. Summary of main results 1. It is sufficient to express an activated form of Toll in hemocytes, but Toll signaling is sufficient and necessary in the fat body to direct hemocyte behavior. All aberrant phenotypes of the Toll10b mutant can be suppressed by knocking down Toll signaling in the fat body. 2. The immune response to L. boulardi is correlated with Toll activation in the fatbody. Discussion 1. The fact that we were able to suppress every phenotype associated with the gain-of-function mutation Tl10b (melanotic nodules, lamellocyte activation, loss of the pattern of sessile cells, and loss of the primary lymph gland lobes) when we suppressed Toll signaling in the fat body suggests that the fat body secretes inducible Toll-dependent gene products into the hemolymph that control hemocyte behavior. In human immunology, cytokines, small molecules that shape the behavior of blood cells, have a long history [209], [210]. In humans, Toll signaling activity induces the production of inflammatory cytokines and interferons in many tissues (reviewed in [211]). Therefore, it seems likely that some of the Toll-dependent genes secreted by the fat body of Drosophila might be cytokines. However, very little is known about cytokines secreted by the fat body that affect hemocyte behavior. Attractive candidates for Toll-dependent cytokines are antimicrobial peptides. But until now to the best of my knowledge, no one has shown that antimicrobial peptides instruct the behavior of hemocytes. We recently published that the infection-inducible peptide edin (elevated during infection) is secreted from fat body and is required for the release of sessile hemocytes after wasp infection [96]. It is not known so far by which mechanism edin instructs hemocyte release. Edin was discovered in an S2 cell screen 24 where it was inducible in a Relish-depended manner in response to heat killed E. coli [212]. Moreover, edin was induced in wntD mutants during L. monocytogenes infection [203]. Notably, wntD is a negative feedback inhibitor under the control of Dorsal/Toll signaling [202]. This suggests that edin expression might be induced by Toll signaling in the fat body. The Drosophila TNF homolog eiger (egr) is expressed in the fat body after infection with S. typhimurium and mediates melanization, antimicrobial peptide secretion, and survival of the Drosophila host [213]. Toll10b is a gain-of-function mutant of Toll that does not require ligand activation [206]. But under normal conditions Toll needs a ligand for its activation. Spätzle (spz) is the only known ligand for the Toll receptor in immunity (reviewed in [214]). Spz is secreted by hemocytes into the hemolymph where it is proteolytically cleaved and then activates Toll signaling in the fat body [36]. More interactions are known where hemocytes send signals to the fat body, however, not all signals induce Toll signaling. The lysosomal protein psidin (phagocyte signaling impaired) is needed for phagosome maturation in plasmatocytes, but also sends an unknown cytokine to the fat body to produce Defensin in response to bacterial infection [215]. Synergistically, nitric oxide and CanA1 (Calcineurin A1) trigger Relish signaling in the fat body in response to feeding of gram-negative bacteria [216]. Septic injury induces the expression of upd3 in hemocytes. Upd3 is secreted from hemocytes and triggers Jak/Stat signaling in the fat body [217]. More complex bidirectional signaling between hemocytes and fat body was described in a tumor model of Drosophila epithelial cells. Polarity loss in tumors triggers the secretion of Pvf1 which induces hemocyte proliferation via its receptor Pvr. Hemocyte-dependent secretion of egr causes tumor cell death and hemocytedependent spz activates the Toll signaling pathway in the fat body resulting in a reduction of tumor burden. The signal the fat body sends to control tumor growth is not yet known [218]. Yet another example for more complex signaling is known for Growth-blocking peptide (Gbp). Gbp is constitutively present in the hemolymph as an inactive form that is activation by proteolysis. Upon various stresses, Gbp activates Metchnikowin expression in an imd- and Jnk-signaling-dependent manner in the fat body [219]. In S2 cells, in addition to activating Metchnikowin expression in an imdand Junk-dependent manner, Gbp stimulates a Ca2+ influx by PLC activation. Elevated Ca2+ levels induce the expression and secretion of Pvf, which in turn activates Erk-signaling within the same cell by binding to its receptor Pvr. Then, Erksignaling promotes cellular immunity while simultaneously inhibiting antimicrobial gene expression [220]. In conclusion, these examples illustrate that cooperation and communication between tissues are important and necessary in facilitating immune signaling. As sentinels, hemocytes recognize dangers and alert the fat body and most likely other tissues by sending signals in the form of cytokines. In response, the fat body and other immune responsive tissues orchestrate the humoral and cellular immune response. The Tl10b mutant might be of help in deciphering the instructive signals the fat body secretes to control hemocyte behavior. 2. The analysis of mutants of the Toll pathway infected by wasps [175] and gene profiling of D. melanogaster after wasp infection [176], [177], [221] illustrate the central role of Toll signaling in the melanotic encapsulation response. The females of some wasp species inject venoms into fly larvae that aim to negatively regulate Toll signaling [197], [198], [222]. We monitored the activity of the Toll signaling pathway with a Drs-GFP reporter [223]. Interestingly, the majority of larvae that had successfully killed the parasite did not express Drs-GFP (Fig. 5 D in paper 4). The analysis of Drs-GFP expression was done 48 h after infection. Possibly, the activity of Toll signaling had abated by that time point. Alternatively, other signaling pathways are able to fill the gap of attenuated Toll signaling. Surprisingly, knocking down Toll signaling components in hemocytes lead to an enhanced encapsulation response early 25 during infection. This result is puzzling because the L. boulardi venoms are aiming to block Toll activity. An explanation might be that Toll signaling in hemocytes has a dampening effect to balance out the immune response. 26 Acknowledgements Thank you! Tack! Kiitos! Danke! A long time has gone by and a huge project has finally come to its end. Many people have contributed to my work, and I owe a big ”thank you” to many people. First, I would like to thank you, Dan, for the opportunity of being able to work in your labs in Umeå and Tampere. And the patience you had with me and my many projects during the years. Soon you will also my ”Doktorvater”, not only Martin´s. Then, I would like to thank my committee, Åsa and Ruth, for some valuable advice during my PhD thesis. I would like to thank all the present (Sajna and Hairu) and past members (JensOla, Jesper, Karin, Svenja, Ingrid, Mazen Michael, MagdaLena, Pia, Calle, AnnaKarin, Karin Ekström, and Anni) of the Hultmark lab in Umeå and the danlab in Tampere. Laura, I am so glad you joined the lab in Tampere. Together we have not only compiled an insane amount of data, but have also done some insanely good research. I hope we will be able to work together in the future. Martin, thank you for the collaboration on our Toll projects. All the best for your fly-bee project. I would also like to say thank you to Tiina and Tuula for the great help you have been in the lab. To all the people at Molecular Biology in Umeå that I still know. It is always nice coming back and feeling welcome. During my time in Tampere, I had the pleasure of supervising a number of very talented students. Thank you, Otto, Siina, Sandra, Johannes, Volker, Ali, and Eveliina for your input into the projects. I would like to say thank you to Mika and the people in his lab, especially Kaisa, Leena-Maija, Susanna, Henna, and Mataleena, for the hospitality I received, when I first came to Tampere. Leena-Maija and Susanna, working with you was nice, and we managed to get some good stuff together. Henna, not so much lab work togther, but the more fun. It was great, spending some long nights together with ”skratanfall”. Eric, I am so glad you never gave up in trying to convince us to using flow cytometry. Alberto, thank you for all the science and carreer related discussions. Bettina, Ana, Tiina, and Päivi, thank you for being good friends here in Tampere. Anna and Dirk, Esther and Arne, Örjan and Annika, andZlatko thank you for all the time we spent together and the fun we had outside of work. Finally, I would like to thank my family for their love and support. 27 References 1. 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