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Genetic screen in Drosophila larvae links ird1 function to Toll signaling in the fat body and hemocyte motility
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine).
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Identifiers
URN: urn:nbn:se:umu:diva-92811OAI: oai:DiVA.org:umu-92811DiVA: diva2:743557
Available from: 2014-09-04 Created: 2014-09-04 Last updated: 2016-09-20Bibliographically approved
In thesis
1. Toll-mediated cellular immune response in Drosophila melanogaster
Open this publication in new window or tab >>Toll-mediated cellular immune response in Drosophila melanogaster
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Insects are amongst the most abundant and diversified multi-cellular organisms on earth. As pollinators of the vast majority of our food crops their socio-economic value is hard to overestimate. Although many pest and pathogens of the honeybee have been known for decades, we still fail to explain the huge losses of honeybee colonies in recent years.At the beginning of my PhD studies, I investigated the effect that senescence and the age-related caste dimorphisms have on two basic parameters of the adult honeybee’s immune system, namely blood cell concentration and the activity of the phenoloxidase cascade. Realizing the limitations of working on an organism for which (at the time) no sequenced genome or molecular tools were available, I switched labs to work on Drosophila melanogaster. The fruit fly has proven to be a particularly useful model system to identify and study genes critical for both the innate immune response itself, as well as the signaling pathways regulating it. For the main part of my thesis, I used the tissue-specific expression of fluorescent markers to visualize segmentally aligned bands of sessile blood cells in the Drosophila larva. This phenotype is disturbed in larvae heterozygote for a gain-of-function mutation in the Toll pathway called Tl10b. In a genetic screen, I scored the ability of genomic mutations to modify the Tl10b loss of bands phenotype. I identified five genomic regions that suppressed the disturbed band pattern of sessile blood cells, and in three of these regions I mapped down this phenotype to single gene level. Two genes are involved in intracellular vesicle trafficking (Rab23 and ird1) and one is activated at the onset of metamorphosis (hdc). To confirm the experimental model, I tested the role of another negative regulator of the Toll pathway. I used tissue specific GAL4 fly lines to express RNAi silencing constructs targeting Gprk2 expression in vivo. This led to an unexpected and novel discovery. Even though blood cells give rise to the most apparent phenotypes in the Tl10b larva, the main source for the immune signal is the fat body. This indicates that besides the humoral response, also in cell based immunity this organ plays a major role. Based on this finding, I could show that the modification of Tl10b blood cell phenotypes caused by loss of ird1 expression are due the role this gene plays in autophagy cell motility. The improved understanding of these basic and evolutionary highly conserved mechanisms will undoubtedly help in fending off infectious disease in both man and honeybees in the future.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2014. 81 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1670
National Category
Biochemistry and Molecular Biology
Research subject
Molecular Biology
Identifiers
urn:nbn:se:umu:diva-92751 (URN)978-91-7601-116-4 (ISBN)
Public defence
2014-09-26, Major Groove, building 6L, molekylärbiologi, Umeå University, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2014-09-05 Created: 2014-09-02 Last updated: 2014-09-08Bibliographically 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
3. Drosophila skeletal muscles regulate the cellular immune response against wasp infection
Open this publication in new window or tab >>Drosophila skeletal muscles regulate the cellular immune response against wasp infection
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Drosophila melanogaster is widely used as a model organism to study the innate immune system because it lacks an adaptive immune response that could mask its innate immune response. The innate immune response of Drosophila primarily consists of humoral and cellular immune responses. The humoral immune response ismediated by antimicrobial peptides, and is induced by bacterial and fungal infections. The cellular immune response is mediated by blood cells (hemocytes), and is induced by bacterial and wasp infection. While the humoral immune response of Drosophila has been studied extensively, the cellular immune response is less well understood.

In this work, I investigated the communication between different signaling pathways and tissues in Drosophila during infection by the parasitic wasp Leptopilina boulardi. I find that JAK/STAT signaling is strongly activated by wasp infection, in both hemocytes and (unexpectedly) larval skeletal muscles. This activation is mediated by the cytokines Upd2 and Upd3, which are secreted from circulating hemocytes. Deletion of upd2 or/and upd3 weakens the wasp-induced activation of JAK/STAT signaling in skeletal muscles and the cellular immune response to wasp infection, leading to reduced encapsulation of wasp eggs and a decrease in the number of circulating lamelloyctes. The suppression of JAK/STAT signaling also significantly weakens the cellular immune response in skeletal muscles, but not in fat bodies and hemocytes. However, the activation of this signaling in skeletal muscles has no obvious effect on the cellular immune response. Together, these results suggest that rather than being uninvolved bystanders, Drosophila skeletal musclesactively participate in cellular immune responses against wasp infection.

To answer how Drosophila larval muscles participate cellular immune response, I min-screened the effects of several immune related signaling pathways in the muscles and the fat body on the cellular immune response. Interestingly, the cellular immune response was only significantly compromised by the suppression ofinsulin signaling in skeletal muscles, in a way that was veryreminiscent of the phenotypes induced by suppressing JAK/STAT signaling in muscles. While wasp infection activates JAK/STAT signaling in muscles, it has the opposite effect on insulin signaling. In addition, I find that insulin signaling in skeletal muscles can positively regulate JAK/STAT signaling. On the other hand, suppression of JAK/STAT signaling in muscles reduces insulin signaling locally in muscles and systemically in the fat body. Suppression of either insulin or JAK/STAT signaling in muscles leads to reductions in glycogen storage in muscles, the trehalose concentration in the hemolymph, and the frequency of feeding behavior. All these results indicate that JAK/STAT and insulin signaling in Drosophila skeletal muscles regulate cellular immune responses via their effects on carbohydrate metabolism. Our findings shed new light on the interactions between diabetes, metabolism, the immune system, and tissue communication.

Place, publisher, year, edition, pages
Umeå: Umeå universitet, 2016. 43 p.
Series
Doctoral thesis / Umeå University, Department of Molecular Biology, 1818Umeå University medical dissertations, ISSN 0346-6612
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-125842 (URN)978-91-7601-508-7 (ISBN)
Public defence
2016-10-11, hörsal E04, NUS 6A–L Biomedicinhuset, Byggnad 6E,, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2016-09-20 Created: 2016-09-20 Last updated: 2016-09-21Bibliographically approved

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Schmid, Martin RAnderl, InesYang, HairuHultmark, Dan

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