Hemese, a hemocyte-specific transmembrane protein, affects the cellular immune response in
            <i>Drosophila</i>

Kurucz, Éva; Zettervall, Carl Johan; Sinka, Rita; Vilmos, Péter; Pivarcsi, Andor; Ekengren, Sophia; Hegedűs, Zoltán; Andó, István; Hultmark, Dan

doi:10.1073/pnas.0436940100

Cited by 149 publications

(153 citation statements)

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“…2 A) based on their differential reactivity with the lectin wheat germ agglutinin (WGA) (33). We confirm the identity of plasmatocytes and lamellocytes (as defined by WGA staining) by costain- ing with the subset-specific P1a and L1b antibodies (21) (Fig. 2B).…”

Section: Detecting Drosophila Hemocytes Through Intracellular Glutathsupporting

confidence: 53%

“…For this purpose, H2, antilamellocyte antibody (L1a), and antiplasmatocyte antibody (P1b) hybridoma supernatants (21) were first fluorescently labeled by using the Alexa 594-Zenon reagents (Molecular Probes) according to the manufacturer's guidelines. Cells were resuspended in 90 l of staining medium, and 10 l of the final antibody solutions were added for a 30-min incubation on ice in the dark.…”

Section: Facs Probes and Staining Proceduresmentioning

confidence: 99%

“…So far, FACS has been applied only once to freshly harvested Drosophila hemocytes, featuring one-parameter analysis of hemolymph (blood surrogate) cells for surface antibody reactivity (21). No FACS analysis of hemocytes from lymph glands, the larval hematopoietic organ (6,14,22), and no cell sorting of fresh hemocytes from either the lymph glands or hemolymph have been reported.…”

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confidence: 99%

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Fluorescence-activated cell sorting (FACS) of Drosophila hemocytes reveals important functional similarities to mammalian leukocytes

Tirouvanziam

Davidson

Lipsick

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Drosophila is a powerful model for molecular studies of hematopoiesis and innate immunity. However, its use for functional cellular studies remains hampered by the lack of single-cell assays for hemocytes (blood cells). Here we introduce a generic method combining fluorescence-activated cell sorting and nonantibody probes that enables the selective gating of live Drosophila hemocytes from the lymph glands (larval hematopoietic organ) or hemolymph (blood equivalent). Gated live hemocytes are analyzed and sorted at will based on precise quantitation of fluorescence levels originating from metabolic indicators, lectins, reporters (GFP and ␤-galactosidase) and antibodies. With this approach, we discriminate and sort plasmatocytes, the major hemocyte subset, from lamellocytes, an activated subset present in gain-of-function mutants of the Janus kinase and Toll pathways. We also illustrate how important, evolutionarily conserved, blood-cell-regulatory molecules, such as calcium and glutathione, can be studied functionally within hemocytes. Finally, we report an in vivo transfer of sorted live hemocytes and their successful reanalysis on retrieval from single hosts. This generic and versatile fluorescence-activated cell sorting approach for hemocyte detection, analysis, and sorting, which is efficient down to one animal, should critically enhance in vivo and ex vivo hemocyte studies in Drosophila and other species, notably mosquitoes.

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Section: Detecting Drosophila Hemocytes Through Intracellular Glutathsupporting

confidence: 53%

Section: Facs Probes and Staining Proceduresmentioning

confidence: 99%

mentioning

confidence: 99%

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Fluorescence-activated cell sorting (FACS) of Drosophila hemocytes reveals important functional similarities to mammalian leukocytes

Tirouvanziam

Davidson

Lipsick

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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“…Furthermore, Notch function is required for a normal cellular response (16). The Hemese gene encodes a hemocyte surface protein, which may play a modulatory role (24).…”

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confidence: 99%

“…To generate a Hemese-GAL4 driver line, we used the PCR to synthesize an Ϸ1-kb-long fragment from the promoter of the Hemese gene (24), by using the following primers with additional EcoRI digestion sites: CGGGATCCATTTGTTGGTAA-TGTCCTCAAGC and GTTTACATAAGTCACTAAGAAT-TCCG. The resulting PCR fragment includes the 5Ј untranslated leader of Hemese and all-intergenic sequence between Hemese and the neighboring gene CG8942.…”

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confidence: 99%

A directed screen for genes involved in Drosophila blood cell activation

Zettervall

Ines

Williams

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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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 hemocytespecific 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 Jak͞Stat 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.

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