Drosophila Hemocytes Research Articles

A role for the Drosophila Toll/Cactus pathway in larval hematopoiesis.

In the Drosophila larva, blood cells or hemocytes are formed in the lymph gland. The major blood cell type, called plasmatocyte, is small, non-adhesive and phagocytic. Plasmatocytes differentiate into adhesive lamellocytes to form multilayered capsules around foreign substances or, in mutant melanotic tumor strains, around self tissue. Mutations in cactus or Toll, or constitutive expression of dorsal can induce lamellocyte differentiation and cause the formation of melanotic capsules. As maternally encoded proteins, Toll, Cactus and Dorsal, along with Tube and Pelle, participate in a common signal transduction pathway to specify the embryonic dorsal-ventral axis. Using the maternal pathway as a paradigm, we investigated if these proteins have additional roles in larval hemocyte formation and differentiation. Analysis of cactus mutants that lack Cactus protein revealed that almost all of these animals have an overabundance of hemocytes, carry melanotic capsules and die before reaching pupal stages. In addition, the lymph glands of cactus larvae are considerably enlarged. The number of mitotic cells in the cactus and TollD hemolymph is higher than that in the wild-type hemolymph. The hemocyte density of mutant Toll, tube or pelle hemolymph is significantly lower than that of the wild type. Lethality of mutant cactus animals could be rescued either by the selective expression of wild-type Cactus protein in the larval lymph gland or by the introduction of mutations in Toll, tube or pelle. Cactus, Toll, Tube and Pelle proteins are expressed in the nascent hemocytes of the larval lymph gland. Our results suggest that the Toll/Cactus signal transduction pathway plays a significant role in regulating hemocyte proliferation and hemocyte density in the Drosophila larva. These findings are discussed in light of similar hematopoietic functions of Rel/I(kappa)B-family proteins in mice.

In vivo regulation of tissue‐specific and LPS‐inducible expression of the Drosophila Cecropin genes

The inducible production of antibacterial cecropins in Drosophila fat body and haemocytes is controlled at the level of transcriptional induction. We demonstrate using germ-line transformation that a short, highly conserved, DNA region, including the insect kappaB motif, is necessary for tissue-specific expression in larvae and adults. Quantitative measurements of reporter gene activity in extracts from transgenic larvae confirmed the requirement of this proximal region for LPS-inducible expression in vivo. Transient expression in a blood cell line indicates the existence of positively acting elements further upstream of the conserved region. Furthermore, our in vivo data suggests that the distal upstream region contains negatively acting element(s).

The expression of MDP-1, a component of Drosophila embryonic basement membranes, is modulated by apoptotic cell death.

Using a novel monoclonal antibody we have studied the expression of a large proteoglycan-type molecule in Drosophila embryos. This molecule is secreted exclusively by migratory, embryonic hemocytes/macrophages and was therefore named MDP-1 for Macrophage-Derived Proteoglycan-1. Expression of MDP-1 begins late during hemocyte differentiation, after these cells have left their birthplace in the head mesoderm. At this time, macrophages are engaged in extracellular matrix deposition and the phagocytosis of cell debris generated by apoptotic events in various parts of the embryo, in particular from the developing central nervous system. Embryos deficient for programmed cell death display a greatly reduced amount of MDP-1 deposition in tissues that normally undergo morphogenetic cell death. This suggests a regulatory role for apoptosis in the terminal differentiation of Drosophila hemocytes. MDP-1 is initially deposited around the developing central nervous system and is later found in basement membrane structures surrounding various other organs, such as the gut, Malpighian tubules and part of the tracheal system. The temporal and localized deposition of MDP-1 suggests that it may play a role in delineating the central nervous system structure during axonogenesis and may participate in the formation of a functional 'blood-brain barrier' in Drosophila.

Helix pomatia lectin and annexin V, two molecular probes for insect microparticles: possible involvement in hemolymph coagulation

Insect hemolymph coagulation involves a complex reaction with contributions from hemocytes and soluble factors. Here we present evidence for the presence of microparticles in the coagulation reaction. These particles are formed by hemocytes in a calcium-dependent process. Both the particles and the remaining cells are labelled by annexin V indicating the presence of phosphatidylserine on the outer membrane. Microparticles are enriched in hemomucin, a surface protein of Drosophila hemocytes that is specifically recognised by a snail ( Helix pomatia) lectin. Hemomucin is shown to bind to lipophorin, a multifunctional hemolymph molecule previously implied in coagulation. Our findings suggest similarities at a biochemical and cellular level between vertebrate blood and insect hemolymph coagulation.

Signals from the IL-1 Receptor Homolog, Toll, Can Activate an Immune Response in a Drosophila Hemocyte Cell Line

The Toll gene encodes an interleukin 1 receptor-like protein that mediates dorsoventral polarity in the Drosophila embryo. The possible involvement of Toll or Toll-like proteins also in the Drosophila immune response was investigated by overexpressing Toll 10B, a constitutively active mutant protein, in the Drosophila blood cell line mbn-2. Induction of the Cecropin A1 ( CecA1) gene, coding for a bactericidal peptide, was used as an indicator for the immune response. Toll 10B was found to increase CecA1 transcription, as detected with a cotransfected CecA1-lacZ reporter gene construct. This effect depends on the presence of a κB-like site in the CecA1 promoter. The endogenous Toll gene is expressed in mbn-2 cells, indicating that this gene may normally play a role in Drosophila blood cells.

Embryonic origin of hemocytes and their relationship to cell death in Drosophila

We have studied the embryonic development of Drosophila hemocytes and their conversion into macrophages. Hemocytes derive exclusively from the mesoderm of the head and disperse along several invariant migratory paths throughout the embryo. The origin of hemocytes from the head mesoderm is further supported by the finding that in Bicaudal D, a mutation that lacks all head structures, and in twist snail double mutants, where no mesoderm develops, hemocytes do not form. All embryonic hemocytes behave like a homogenous population with respect to their potential for phagocytosis. Thus, in the wild type, about 80-90% of hemocytes become macrophages during late development. In mutations with an increased amount of cell death (knirps; stardust; fork head), this figure approaches 100%. In contrast, in these mutations, the absolute number of hemocytes does not differ from that in wild type, indicating that cell death does not 'induce' the formation of hemocytes. Finally, we show that, in the Drosophila embryo, apoptosis can occur independently of macrophages, since mutations lacking macrophages (Bicaudal D; twist snail double mutants; torso4021) show abundant cell death.

Hemocyte encapsulation of streptococci in Drosophila

Electron micrographs of infectious bacterial cells encapsulated by hemocytes in Drosophila suggest that bacterial lysis within the capsular confines may be the mode of the hemocyte defense reaction rather than intracellular phagocytic destruction.