Signaling pathways involved in hematopoiesis and immunity in Drosophila Erika Bach email@example.com Goals of lecture • Hematopoiesis (blood cell development) in embryos and larval stages • Immunity (responses to foreign organisms and pathogens) in larvae and adults Drosophila life cycle • Four major stages – Embryo – Larva (3 instars) – Pupa – Adult • Short - 9 days long • Easily observed • Powerful genetics available Functions of blood cells • Phagocytose apoptotic cells during development • Tissue remodeling • Immune responses – phagocytosis – arrest microbial growth – secrete anti-microbial peptides Why do flies need blood cells? Early embryonic blood cell development A. Stage 5: hemocytes arise from the hemocyte anlagen in embryonic mesoderm B. Stage 11 – 700 prohemocytes appear 2 hrs after gastrulation (st.10) – express the GATA transcription factor serpent – migrate throughout the embryo – differentiate into: plasmatocytes and crystal cells Late embryonic blood cell development C. Stage 17 Plasmatocytes • are induced to become macrophages • phagocytose dying cells • participate in immune responses? Crystal cells • unknown function in the embryo Major types of embryonic blood cells • Plasmatocytes – comprise 90% of embryonic hemocytes – acquire phagocytic capabilities after expression of Croquemort, CD36, a scavenger receptor – act as macrophages in tissue remodeling and phagocytose dead or dying cells at the end of embryogenesis • Crystal cells – >10% embryonic hemocytes – cytoplasmic crystalline inclusions – unknown function Marker of embryonic hemocytes • Prohemocytes express Serpent (srp) - srp is required for embryonic hematopoiesis and formation of the fat body (fly “liver”) - srp is highly homologous to hematopoietic GATA factors • Plasmatocytes express glial cells missing (gcm) - Gcm and its mammalian homologs have all been shown to bind the same binding site sequence with similar affinities • Crystal cells express Lozenge (Lz) - Lz contains a Runt domain - Lz is highly homologous to AML1 protein aml1 is required for definitive hematopoiesis aml1 locus is the most frequent target of chromosome rearrangement in acute myeloid leukemia. GATA Factors: Structure and function AML/Runx proteins and their functions srp mutants lack prohemocytes gcm and gcm2 are required for transition from plasmatocyte to macrophage gcm and gcm2 are required for: • proliferation of plasmatocyte precursors • the expression of Croquemort • the ability of plasmatocytes to convert into macrophages Lozenge is required for crystal cell development Permissive temp Restrictive temp embryo larva Relationships between Srp, Lz and Gcm • In srp mutants, lz is not expressed • Srp and Gcm do not co-localize in a group of cells presumed to be CCP • Expression of Lz and Gcm is mutually exclusive • Mis-expression of Gcm in CCP results in croquemort expression • Gcm-expressing CCP have altered morphology, consistent with change in cell fate Lineage of embryonic hemocytes Evolutionary conservation of hematopoietic developmental programs? GATA-1 KO mice have no red blood cells Mammalian hematopoiesis Larval hematopoiesis • the primary organ of larval hematopoiesis is the lymph gland • prohemocytes in the lymph gland – self-renew and differentiate into specific types of hemocytes – hemocytes are released into the circulatory system, called the hemolymph – the circulatory system in flies is open, and the internal organs are surrounded by hemolymph Post-embryonic hematopoiesis Larval hemocytes • plasmatocytes – 90% of larval hemocytes, small, non-adhesive cells – under immune challenge or pupariation, they become activated, increase in number and in phagocytosis and secretion • lamellocytes – Are induced to differentiate following immune challenge – V. large, flat cells, separate lineage from plasmatocytes – encapsulate objects too large to be phagocytosed which are then melanized by crystal cell • crystal cell – 5% of larval hemocytes – have crystalline-like inclusion in their cytoplasm – separate lineage from plasmatocytes as mutants that lack crystal cells, e.g. lz mutants, have plasmatocytes Signaling pathways involved in larval hematopoiesis: JAK/STAT pathway • JAK/STAT pathway is essential for mammalian blood cell development • hop and stat92E are involved in hematopoiesis The Drosophila JAK/STAT pathway JAK/STAT pathway in fly hematopoiesis • gain-of-function mutations hopTum-l and hopT42 encode hyperactive kinases • at permissive and restrictive temps, they exhibit over- proliferation and precocious differentiation of hemocytes – In 2nd instar hopTum-l and hopT42 larvae, lamellocytes are 10-15% of the total hemocytes, compared to <1% in WT – By 3rd instar, lamellocytes in hopTum-l larvae are 50-75% of the total hemocytes, compared with <5% in WT – plasmatocytes frequently have abnormal intracellular features – lamellocytes in hopTum-l and hopT42 larvae aggregate into masses that often become melanized, the so-called “melanotic tumors” Melanotic tumor • appears as a black mass • consists of at least two cell types, plasmatocytes that are encapsulated by lamellocytes • the aggregate is melanized, presumably by activation of the prophenoloxidase cascade in crystal cells • strongly correlated with lethality Signaling pathways involved in larval hematopoiesis: Toll/NFkB pathway • Loss-of-function mutants (Tl, tb, pll) have reduced hemocytes • cac l-o-f mutants and Tl g-o-f mutants (Tl10B) have increased hemocytes and melanotic tumors (like hopTum-l) • JAK/STAT and Tl pathways regulate genes in blood cell proliferation/differentiation Signaling pathways in involved larval hematopoiesis: Notch • Notch l-o-f mutants have in decreased crystal cells • Over-expression of Notch induces differentiation of crystal cells • Notch function is necessary for lamellocyte proliferation upon parasitization • Notch does not play a role in the differentiation of the plasmatocytes Innate immunity in flies and man What is innate immunity? • Innate immunity concerns the detection of constitutive and conserved motifs in pathogens and the response to it. • In contrast, in adaptive immunity foreign organisms are not directly sensed and acquires 4-7 days for clonal expansion T and B cells. (Mammals have acquired immunity, flies do not.) • Thus, the innate immune system keeps us alive while the adaptive immune response is gearing up and also participates directly in shaping the adaptive immune response. An important observation: the Tl pathway is involved in immunity in Drosophila • B. Lemaitre, J. Hoffmann and colleagues showed definitely in 1996 that mutations in the Tl gene resulted in susceptibility to fungal infection • This was extremely exciting because until then the Tl pathway had been very extensively studied as the major dorsal-ventral patterning pathway in the embryo. The Toll pathway in mammals linked innate and acquired immunity! • Until 1997, immunologists had thought that innate immunity primary function was to keep you alive until adaptive immunity irradicated the pathogen • However, Medzhitov and Janeway showed that human Toll receptors were a link between innate and adaptive immunity. • They cloned and characterized a human homologue of the Drosophila Toll and showed that a constitutively active human Toll induces: – the activation of NF-kB – the expression of NF-kB-controlled genes for the inflammatory cytokines IL-1, IL-6 and IL-8 – the expression of the costimulatory molecule B7.1, which is required for the activation of naive T cells. How do flies mount immune responses? • Humoral response: the fat body produces antimicrobial peptides which are released into the circulatory system • Cellular response: hemocytes expand and differentiate and participate in phagocytosis and encapsulation • Induction of proteolytic cascades (leading to melanization and coagulation) and production of signaling molecules Innate immunity in Drosophila PAMPs and PRRs • Pathogen-associated molecular patterns (PAMPs) are not virulence factors – Produced only by microbes – Invariant between class of microbes – Essential for microbial survival • Pattern-recognition receptors (PRRs) (in host) – Sense PAMPs – Cannot distinguish pathogen vs. non-pathogen – Functions include: • Opsonization and phagocytosis • Activation of complement • Activation of pro-inflammatory signaling cascases Antimicrobial peptides (AMPs) • In response to septic injury, AMPs are produced rapidly and massively in the larval fat body (and also by hemocytes) and are released immediately into the hemolymph. • AMP genes have kB sites in their promoters, and thus are regulated by NFkB/Rel proteins that are activated in response to infection. • What other pathways are involved in the induction of proteolytic cascades or AMPs? Comparison of AMPs NFkB-like proteins in Drosophila • Dorsal (Dl) is involved in dorso-ventral patterning in the embryo (not immune responses) • Dorsal-type immune factor (Dif) is mainly involved in the induction of AMP genes in response to fungal and gram-positive bacterial challenge • Relish (Rel) regulates the induction of peptides active against Gram-negative bacteria Current view of larval immunity (c) Stress- response upd3 Dome Responses to fungi and gram+ bacteria • Both require the Tl pathway • In mice, Tl4 acts as a direct sensor of microbial compounds (eg LPS) • However, in flies Tl is not a direct sensor. In the embryo, Spz is cleaved by serine proteases from a “pro” form. In immunity, a proteolytic cascade activates Tl ligand Spaetzle • In immunity, the drosomycin gene is constitutively on in mutants for the necrotic gene (a serine protease inhibitor - serpin) How does this upstream protease cascade sense the pathogen in flies? • Fungi - not known • Gram + bacteria - peptidoglycan recognition protein (PGRP) – Flies have 13 PGRP genes and several are upregulated in response to septic injury. – PGRP-SA (semmelweis) is required for activation of Tl after exposure to Gram + bacteria As an aside…what about the other 8 Tl genes in flies? • Flies have 9 Toll receptor genes • Do they all function in immunity? Responses to gram negative bacteria • Immunity involves the immune deficient (imd) pathway. • Ligand is gram-negative peptidoglycan Diaminopimelic acid (DAP) • Receptor may be PGRL-LC Stress-response in Drosophila • Upon tissue damage, what are the signaling pathways that are activated to restore homeostasis? • To be discussed in detail on Thursday (Agaisse et al, Dev Cell, 2003) Genetic screens for immune function ird7 mutants do not induce Diptericin or activate Relish in response to Gram negative bacteria UAS-GAL4 tissue specific expression X UAS-GFP Promoter-GAL4 ey-GAL4/ UAS-GFP PGRP rescues Dipt-lacZ induction in response to E. coli in ird7 mutants Conclusions I • Evolutionary conversation of embryonic (and larval?) developmental programs of hematopoiesis - – key players serpent, a GATA factor and lozenge, an aml- like factor – Future work: what controls these factors in flies? Parallel story in vertebrates? • Tl and JAK-STAT pathways control hemocyte differentiation in larva - – Future work: what are the ligands and receptors? How are the Tl and JAK-STAT pathways inter-connected? Conclusions: II Fungal and Gram+ immunity Conclusions III: Gram - immunity Selected references • Lebestky T, Chang T, Hartenstein V, Banerjee U. Specification of Drosophila hematopoietic lineage by conserved transcription factors. Science. 2000 Apr 7;288(5463):146-9 • Hoffmann JA, Reichhart JM., Drosophila innate immunity: an evolutionary perspective.Nat Immunol. 2002 Feb;3(2):121- 6. Review • Lanot R, Zachary D, Holder F, Meister M. Postembryonic hematopoiesis in Drosophila., Dev Biol. 2001 Feb 15;230(2):243-57. • Tzou P, De Gregorio E, Lemaitre B. How Drosophila combats microbial infection: a model to study innate immunity and host-pathogen interactions.Curr Opin Microbiol. 2002 Feb;5(1):102-10. • Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell. 1996 Sep 20;86(6):973-83. • Medzhitov R.Toll-like receptors and innate immunity. Nat Rev Immunol. 2001 Nov;1(2):135-45. • Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity.Nature. 1997 Jul 24;388(6640):394-7. • Choe KM, Werner T, Stoven S, Hultmark D, Anderson KV. Requirement for a peptidoglycan recognition protein (PGRP) in Relish activation and antibacterial immune responses in Drosophila. Science. 2002 Apr 12;296(5566):359-62. • Wu LP, Choe KM, Lu Y, Anderson KV. Drosophila immunity: genes on the third chromosome required for the response to bacterial infection., Genetics. 2001 Sep;159(1):189-99. • Ligoxygakis P, Pelte N, Hoffmann JA, Reichhart JM. Activation of Drosophila Toll during fungal infection by a blood serine protease. Science. 2002 Jul 5;297(5578):114-6. • Gottar M, Gobert V, Michel T, Belvin M, Duyk G, Hoffmann JA, Ferrandon D, Royet J. The Drosophila immune response against Gram-negative bacteria is mediated by a peptidoglycan recognition protein. Nature. 2002 Apr 11;416(6881):640-4 • Cantor AB, Orkin SH. Transcriptional regulation of erythropoiesis: an affair involving multiple partners. Oncogene. 2002 May 13;21(21):3368-76. • Westendorf JJ, Hiebert SW. Mammalian runt-domain proteins and their roles in hematopoiesis, osteogenesis, and leukemia. J Cell Biochem. 1999;Suppl 32-33:51-8.
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