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A directed screen for genes involved in Drosophila blood cell activation
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine). (Dan Hultmark)
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine). (Dan Hultmark)
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine). (Dan Hultmark)
Umeå University, Faculty of Medicine, Umeå Centre for Molecular Pathogenesis (UCMP) (Faculty of Medicine). (Ruth Palmer)
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2004 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 101, no 39, 14192-14197 p.Article in journal (Refereed) Published
Abstract [en]

An attack by a parasitic wasp activates a vigorous cellular immune response in Drosophila larvae. This response is manifested by an increased number of circulating cells, the hemocytes, and by the appearance of a specialized class of hemocyte, the lamellocytes, which participate in the encapsulation and killing of the parasite. To study the molecular mechanisms of this response, we have overexpressed different genes in the hemocytes, by using the GAL4-upstream activating sequence system and a hemocyte-specific Hemese-GAL4 driver. Multiple transgenes were tested, representing several important signaling pathways. We found that the proliferation response and the activation of lamellocyte formation are independent phenomena. A drastic increase in the number of circulating hemocytes is caused by receptor tyrosine kinases, such as Egfr, Pvr, and Alk, as well as by the downstream signaling components Ras85D and pointed, supporting the notion that the Ras-mitogen-activated protein kinase pathway regulates hemocyte numbers. In the case of Pvr and Alk, this phenotype also is accompanied by lamellocyte formation. By contrast, constitutively active hopscotch and hemipterous give massive activation of lamellocyte formation with little or no increase in total hemocyte numbers. This finding indicates that both the Jak/Stat and the Jun kinase pathways affect lamellocyte formation. Still other signals, mediated by aop(ACT), Toll(10b), and Rac1 expression, cause a simultaneous increase in lamellocyte and total cell numbers, and the same effect is seen when WNT signaling is suppressed. We conclude that the activation of a cellular response is complex and affected by multiple signaling pathways.

Place, publisher, year, edition, pages
Washington: National Academy of Sciences , 2004. Vol. 101, no 39, 14192-14197 p.
Keyword [en]
Animals, Drosophila/*genetics/*immunology/parasitology, Drosophila Proteins/genetics/metabolism, Female, Gene Expression, Hemocytes/enzymology/immunology/parasitology/*physiology, Larva/immunology/metabolism/parasitology, Male, Phenotype, Receptor Protein-Tyrosine Kinases/metabolism, Recombinant Fusion Proteins/genetics/metabolism, Signal Transduction, Transgenes/*genetics, Wasps/immunology, beta-Galactosidase/genetics, ras Proteins/metabolism
National Category
Infectious Medicine
Identifiers
URN: urn:nbn:se:umu:diva-17054DOI: 10.1073/pnas.0403789101ISI: 000224211400042PubMedID: 15381778OAI: oai:DiVA.org:umu-17054DiVA: diva2:156727
Available from: 2007-10-28 Created: 2007-10-28 Last updated: 2015-08-13Bibliographically approved
In thesis
1. Signaling pathways in the activation and proliferation of Drosophila melanogaster blood cells
Open this publication in new window or tab >>Signaling pathways in the activation and proliferation of Drosophila melanogaster blood cells
2005 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The larva of the fruit fly Drosophila melanogaster is an excellent model to study the molecular control of innate cellular immune responses. Cellular responses take place, and can be studied, following infestation of the wasp Leptopilina boulardi. This response includes proliferation and activation (differentiation) of the blood cells (hemocytes). In a successful anti-parasitic response, an immune-induced lineage of hemocytes, the lamellocytes, forms a cellular capsule covering and killing the foreign intruder. I will in this thesis present data about the finding and characterization of a novel marker that is expressed specifically in the hemocytes, the Hemese gene. I furthermore describe the construction of a useful tool, the transgenic Hemese-Gal4 fly, which enables blood cell specific expression of any gene of interest. By using the Hemese-Gal4 fly in a directed screen, I have found that a surprisingly large number of genes, that in turn are members of seemingly diverse signaling pathways, are able to induce a cellular response. In many cases their expression is also associated with a blood cell tumor phenotype. Overexpression of certain genes, such as hopscotch (a Drosophila Jak homologue) and hemipterous (a c-jun kinase kinase) lead to the formation of lamellocytes. Other genes may control the cell number, such as Egfr and Ras, as their expression produced a massive in increase the numbers of hemocytes. A third group of genes, including, e.g. Alk, Rac1 and Pvr give a mixed response, promoting both hemocyte proliferation and activation. Surprisingly, the suppression of WNT signaling in hemocytes lead to hemocyte activation. In one case, with a UAS-Pvr dominant negative construct, we observe a reduction of the circulating blood cells in uninfested larva. The expression of DN-Pvr additionally contributes to reduce encapsulation rates in larvae subjected to Leptopilina infestation. In conclusion: the control of blood cells in larval hematopoiesis, and during parasitic wasp attacks, is complex and may involve multiple pathways. In a broader sense, the gene functions found in the directed screen may have implications also for understanding the molecular control of mammalian myeloid lineage blood cells.

Place, publisher, year, edition, pages
Umeå: Umeå centrum för molekylär patogenes (UCMP), 2005. 49 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 965
Keyword
innate immunity, cellular immunity, molecular signaling
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-513 (URN)91-7305-876-9 (ISBN)
Public defence
2005-05-13, Hörsal Betula, by 6 M, Umeå, 09:00
Opponent
Available from: 2005-04-25 Created: 2005-04-25 Last updated: 2011-04-08Bibliographically approved
2. Activation of the Cellular Immune Response in Drosophila melanogaster Larvae
Open this publication in new window or tab >>Activation of the Cellular Immune Response in Drosophila melanogaster Larvae
2015 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2015. 41 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1741
Keyword
Drosophila melanogaster, immunity, blood cells, hematopoiesis, flow cytometry, in vivo imaging, genetic screens, Tl10b, fat body, Toll signaling
National Category
Immunology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-106981 (URN)978-91-7601-317-5 (ISBN)
Public defence
2015-09-07, Major Groove, Byggnad 6L, Umeå, 12:30 (English)
Opponent
Supervisors
Available from: 2015-08-17 Created: 2015-08-13 Last updated: 2015-08-13Bibliographically approved

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Anderl, InesWilliams, MichaelPalmer, RuthHultmark, Dan

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