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Signaling pathways in the activation and proliferation of Drosophila melanogaster blood cells
Umeå University, Faculty of Science and Technology, Umeå Centre for Molecular Pathogenesis (UCMP).
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 [en]
innate immunity, cellular immunity, molecular signaling
Research subject
Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-513ISBN: 91-7305-876-9 (print)OAI: oai:DiVA.org:umu-513DiVA: diva2:143659
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
List of papers
1. Hemese, a hemocyte-specific transmembrane protein, affects the cellular immune response in Drosophila.
Open this publication in new window or tab >>Hemese, a hemocyte-specific transmembrane protein, affects the cellular immune response in Drosophila.
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2003 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 100, no 5, 2622-7 p.Article in journal (Refereed) Published
Abstract [en]

We have identified a previously undescribed transmembrane protein, Hemese, from Drosophila melanogaster blood cells (hemocytes), by using a monoclonal pan-hemocyte antibody. Heavy glycosylation is suggested by the heterogeneous size distribution, ranging between 37 and 70 kDa. Hemese expression is restricted to the cell surfaces of hemocytes of all classes, and to the hematopoietic organs. The sequence of the corresponding gene, Hemese (He), predicts a glycophorin-like protein of 15 kDa, excluding an N-terminal signal peptide, with a single hydrophobic transmembrane region. The extracellular region consists mainly of Ser/Thr-rich sequence of low complexity, with several potential O-glycosylation sites. Hemese contains phosphotyrosine and the cytoplasmic region has potential phosphorylation sites, suggesting an involvement in signal transduction. Depletion of Hemese by RNA interference has no obvious effect under normal conditions, but the cellular response to parasitic wasps is much enhanced. This finding indicates that Hemese plays a modulatory role in the activation or recruitment of the hemocytes.

Keyword
Amino Acid Sequence, Animals, Blotting; Western, Cell Membrane/*metabolism, Cell Separation, Cloning; Molecular, Cytoplasm/metabolism, DNA; Complementary/metabolism, Drosophila, Drosophila Proteins/*chemistry/*physiology, Flow Cytometry, Gene Library, Glycosylation, Green Fluorescent Proteins, Hemocytes/*metabolism, Hybridomas, Immunohistochemistry, Luminescent Proteins/metabolism, Membrane Proteins/*chemistry/*physiology, Molecular Sequence Data, Peptides/chemistry, Phagocytosis, Phosphorylation, Precipitin Tests, Protein Structure; Tertiary, RNA/metabolism, RNA Interference, Signal Transduction, Tyrosine/metabolism
Identifiers
urn:nbn:se:umu:diva-17060 (URN)10.1073/pnas.0436940100 (DOI)12598653 (PubMedID)
Available from: 2007-10-28 Created: 2007-10-28 Last updated: 2017-12-14
2. A directed screen for genes involved in Drosophila blood cell activation
Open this publication in new window or tab >>A directed screen for genes involved in Drosophila blood cell activation
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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
Keyword
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:nbn:se:umu:diva-17054 (URN)10.1073/pnas.0403789101 (DOI)000224211400042 ()15381778 (PubMedID)
Available from: 2007-10-28 Created: 2007-10-28 Last updated: 2017-12-14Bibliographically approved
3. Pvf2/Pvr signaling in proliferating and activated Drosophila hemocytes.
Open this publication in new window or tab >>Pvf2/Pvr signaling in proliferating and activated Drosophila hemocytes.
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-4523 (URN)
Available from: 2005-04-25 Created: 2005-04-25 Last updated: 2010-01-13Bibliographically approved

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