Drosophila has recently become a powerful model organism for studies of innate immunity. The cellular elements of innate immunity in Drosophila, the hemocytes, have been characterized by morphological criteria, molecular markers, and cell-type-specific immunological markers. Here we suggest that an MiET1 GFP-reporter element insertion in the untranslated region of a gene (l1-atilla) - expressed in a subset of hemocytes, the lamellocytes - allows in vivo investigations of lamellocyte differentiation and facilitates genetic screens

Brand

Braun

Carton

Evans

Goto

Hartenstein

Hoffmann

Hultmark

Konrad

Kurucz

Lanot

Metaxakis

Márkus

Zettervall

English

Honti, Viktor

Kurucz, Éva

Csordás, Gábor

Laurinyecz, Barbara

Márkus, Róbert

Andó, István

Repository of the Academy's Library

Authors: Honti V., Kurucz É., Csordás G., Laurinyecz B., Márkus R., Andó I.Title: In vivo detection of lamellocytes in Drosophila melanogaster.Journal: Immunology lettersYear:  2009Vol: 126 Page: 83-84DOI: 10.1016/j.imlet.2009.08.004PMID: 19695290NOTICE: this is the author’s version of a work that was accepted for publication in Immunology Letters. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Immunology Letters vol 126, Issue 1-2, 2009. DOI: 10.1016/j.imlet.2009.08.004 In vivo detection of lamellocytes in Drosophila melanogaster Keywords: hemocyte, in vivo, lamellocyte, Drosophila, differentiation Sir,In  both  vertebrates  and  invertebrates  the  blood  cells  undergo  differentiation  in  spatially separated  organs,  in  a  characteristic  time  sequence  [1-5].  Recently  Drosophila became  a powerful model organism for studying blood cell development and cellular events of innate immunity.  The  cellular  elements  of  innate  immunity,  the  blood  cells  or  hemocytes  in Drosophila,  have  been  characterized  by  morphological  criteria  and  molecular  markers, including transcription factors [6,7] and cell-type specific immunological markers [8-10]. In Drosophila, at least three main classes of hemocytes can be discerned. The predominant class comprises small round cells with a phagocytic capacity, the plasmatocytes. A second class, the crystal cells,  distinguished by pronounced crystal  like inclusions in the cytoplasm, are involved in melanin deposition in wounds and around foreign objects. Finally, a class of large flat  cells,  the  lamellocytes,  appears  when  the  larvae  are  infected  by  parasitoid  wasps. Lamellocytes  participate  in  the encapsulation  of  parasites  and differentiate  in  response  to mechanical assault and their development is finely regulated [11, 12, 5]. Hemocytes are distributed in three main hematopoietic compartments in the larva, the lymph gland,  the  sessile  tissue  and  the  circulation.  The  understanding  of  molecular  events  of hemocyte development in these compartments and the definition of signalling pathways that lead to differentiation and the immune response have been facilitated by genetic screens in combination with functional tests and monitoring of hemocyte subsets. This analysis has been helped by the availability of reporter-gene constructs expressing marker molecules, including fluorescent  proteins,  in  vivo.  The  constructs  make  possible  to  analyze  and  follow  the hemocyte  development  in  vivo by  expressing  fluorescent  proteins  in  a  cell-type  specific manner.  Plasmatocytes,  lamellocytes  and  crystal  cells  can  be  visualized  by  appropriate constructs [11, 13-15];  however, lamellocyte specific marker for  in vivo use has not been described yet. Recently we have defined a molecular marker for lamellocytes as Atilla-L1, and determined the localization of its gene in the genome [Laurinyecz et al., unpublished]. Database analysis revealed that an insertion of a MiET1 element [16] in this gene is available.  The element contains a hsp70 promoter controlled  GAL4 gene, and an EGFP marker gene driven by the 3xP3  promoter.  The  Mi{ET1}CG6579[MB03539] insertion  is  located  in  the  atilla gene (CG6579) [Laurinyecz et al., unpublished] at position 2L:12,177,639 in the ‘plus’ orientation in the 5’ untranslated region. Our analysis revealed that the stock carrying this insertion is suitable for in vivo detection of lamellocytes in immune-challenged larvae (Fig. 1A, arrows) as well as in hemocyte overproducing mutants. The use of a panel of lamellocyte specific markers [10] showed that the expression of GFP is specific for/to? lamellocytes. The 3xP3 promoter also shows an expression pattern similar to that of the eyeless driver, which appears in the eye discus, brain, gut and anal plate (Fig. 1A, arrowheads) showing up as a background fluorescence appearing in all stocks carrying the  MiET1 element [16]. The insertion blocks the  expression  of  the  Atilla  protein  encoded  by  the  atilla gene,  but  does  not  affect  the expression of other, independent lamellocyte-specific markers [10], as L2 and L6 (Fig. 1B,C). These  results  confirm  that  the  insertion  allows  in  vivo investigation  of  lamellocyte differentiation as well as helps genetic screens with the aim of dissecting signaling pathways of hemocyte development. We also found that the GAL4 gene in this insertion is inactive. This however allows the use of this insertion with independent GAL4 drivers in combination with UAS-RFP reporter construct thus making possible double-labelling of hemocyte populations in vivo with a clear distinction of lamellocytes. Acknowledgement:  Hungarian National  Research Fund Grants,  OTKA K-68830 and NK-78024 supported this research and the technical help of Szilvia Tápai is acknowledged. References[1] Hultmark D. Insect immunology. Ancient relationships. Nature 1994;367:116-7.[2] Hoffmann JA, Reichhart JM. Drosophila innate immunity: an evolutionaryperspective. Nature Immunol 2002;3:121-6.[3] Hartenstein V. Blood cells and blood cell development in the animal kingdom. Annu Rev Cell Dev Biol 2006;22:677-712. Review.[4] Lanot  R,  Zachary  D,  Holder  F,  Meister  M.  Postembryonic  hematopoiesis  in Drosophila. Dev Biol 2001;230:243-57.[5] Márkus  R,  Laurinyecz  B,  Kurucz  E,  Honti  V,  Bajusz  I,  Sipos  B  et  al.  Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster. Proc Natl Acad Sci USA 2009;106:4805-9.[6] Evans CJ, Hartenstein V, Banerjee U. Thicker than blood: conserved mechanisms in Drosophila and vertebrate hematopoiesis. Dev Cell 2003;5:673-90. Review.[7] Evans CJ, Banerjee U. Transcriptional regulation of hematopoiesis in Drosophila.Blood Cells Mol Dis 2003;30:223-8. Review.[8] Kurucz E, Zettervall CJ, Sinka R, Vilmos P, Pivarcsi A, Ekengren S et al. Hemese, a hemocyte-specific  transmembrane  protein,  affects  the  cellular  immune  response  in Drosophila. Proc Natl Acad Sci USA 2003;100:2622-7.[9] Kurucz E, Márkus R, Zsámboki J, Folkl-Medzihradszky K, Darula Z, Vilmos P et al. Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes. Curr Biol 2007;17:649-54.[10] Kurucz E, Váczi B, Márkus R, Laurinyecz B, Vilmos P, Zsámboki J et al. Definition of  Drosophila  hemocyte  subsets  by  cell-type  specific  antigens.  Acta  Biol  Hung  2007;58 Suppl:95-111.[11] Zettervall CJ, Anderl I, Williams MJ, Palmer R, Kurucz E, Ando I et al. A directed screen for genes involved in Drosophila blood cell activation.  Proc Natl Acad Sci U S A 2004;101:14192-7.[12] Márkus R, Kurucz E, Rus F, Andó I. Sterile wounding is a minimal and sufficient trigger  for  a  cellular  immune  response  in  Drosophila  melanogaster.  Immunol  Lett 2005;101:108-11.[13] Goto  A,  Kadowaki  T,  Kitagawa  Y.  Drosophila  hemolectin  gene  is  expressed  in embryonic  and  larval  hemocytes  and  its  knock  down  causes  bleeding  defects.  Dev  Biol 2003;264:582-91.[14] Braun A, Lemaitre B, Lanot R, Zachary D, Meister M. Drosophila immunity: analysis of larval hemocytes by P-element-mediated enhancer trap. Genetics 1997;147:623-34. [15] Brand AH, Perrimon N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 1993;118:401-15.[16] Metaxakis A, Oehler S, Klinakis A, Savakis C. Minos as a genetic and genomic tool in Drosophila melanogaster. Genetics 2005;171:571-81.Viktor HontiÉva KuruczGábor CsordásBarbara LaurinyeczRóbert MárkusIstván Andó*Institute of Genetics, Biological Research CenterTemesvári krt. 62, 6726 Szeged Hungary*:Tel.: +36 62599677; fax: +36 62433503.E-mail address: ando@brc.huLegend to Fig. 1 Lamellocytes in the Mi{ET1}CG6579[MB03539] larva (A)  GFP  expressing  lamellocytes  are  visible  through  the  cuticule  of  the  wasp  infested Mi{ET1}CG6579[MB03539] larva. Arrows are pointing at lamellocytes, arrowheads point to organs showing background fluorescence characteristic  for the  MiET1 element  [16] (scale bar:  100µm).  (B,C)  The  GFP  expression  overlaps  with  the  L2  (B)  and  L6  (C)  antigen expression in the hemocytes of homozygous  Mi{ET1}CG6579[MB03539]; l(3)mbn1 larvae. Lamellocytes  are  visualized  by  indirect  immunofluorescence.  The  GFP  expression  in lamellocytes is enhanced by rabbit anti-GFP staining and anti-rabbit Alexa-488 (green). The expression of lamellocyte specific antigens is detected with mouse anti-L2, anti-L6 and anti-mouse Alexa-568 (red). Arrows are pointing at/to ? double labelled lamellocytes (scale bars : 20µm).

A directed screen for genes involved in Drosophila blood cell activation.

Blood cells and blood cell development in the animal kingdom. Annu Rev Cell Dev Biol

Definition of Drosophila hemocyte subsets by cell-type specific antigens.

Drosophila hemolectin gene is expressed in embryonic and larval hemocytes and its knock down causes bleeding defects.

Drosophila immunity: analysis of larval hemocytes by P-element-mediated enhancer trap.

Drosophila innate immunity: an evolutionary perspective.

Hemese, a hemocyte-specific transmembrane protein, affects the cellular immune response in Drosophila.

Insect immunology. Ancient relationships.

Postembryonic hematopoiesis in Drosophila.

Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster.

Sterile wounding is a minimal and sufficient trigger for a cellular immune response in Drosophila melanogaster.

Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.

Thicker than blood: conserved mechanisms in Drosophila and vertebrate hematopoiesis. Dev Cell

In vivo detection of lamellocytes in Drosophila melanogaster.

Abstract

Similar works

Full text

Available Versions

Repository of the Academy's Library

Crossref