Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster

Abstract

The blood cells, or hemocytes, in Drosophila participate in the immune response through the production of antimicrobial peptides, the phagocytosis of bacteria, and the encapsulation of larger foreign particles such as parasitic eggs; these immune reactions are mediated by phylogenetically conserved mechanisms. The encapsulation reaction is analogous to the formation of granuloma in vertebrates, and is mediated by large specialized cells, the lamellocytes. The origin of the lamellocytes has not been formally established, although it has been suggested that they are derived from the lymph gland, which is generally considered to be the main hematopoietic organ in the Drosophila larva. However, it was recently observed that a subepidermal population of sessile blood cells is released into the circulation in response to a parasitoid wasp infection. We set out to analyze this phenomenon systematically. As a result, we define the sessile hemocytes as a novel hematopoietic compartment, and the main source of lamellocytes.

Fig. 1.

Morphology and phenotype of Hemese-GFP-expressing sessile hemocytes in parasitized larvae. (A) The sessile hemocytes exhibit a banded pattern in third instar control larvae. The picture shows the pattern at the posterior end of the larva (11). (B) The banded pattern of the sessile hemocytes disappears 72 h after infection. (C–F) Immunostaining of the sessile hemocytes (C and E) uninfected, and (D and F) wasp-infected larva; (C and D) plasmatocytes and (E and F) lamellocytes are visualized with anti-NimC1 (red) or anti-L1 (red) staining, respectively; (D) 72 h, (F) 48 h after infection. The arrows indicate the NimC1 or L1-expressing hemocytes, whereas the arrowheads point to hemocytes that do not express these antigens. [Scale bars: A and B (100 μm) and C–F (20 μm).].

Fig. 2.

Box-and-whiskers plot of hemocyte counts in the sessile (Ses) and circulating (Circ) compartments, 72 h after parasitic wasp infection. (A) Total number of Hemese-GFP-expressing hemocytes per larva in circulation and in the sessile tissue. In this experiment, 349 cells per larva (uninfected 424 minus infected 75, median values) disappeared from the sessile tissue, compared with 331 (infected 1,025 minus uninfected 694) that appeared in circulation. (B) Number of NimC1 negative and (C) NimC1 positive sessile hemocytes. (D) Number of L1-expressing sessile and circulating hemocytes. C, uninfected controls; I, infected larvae.

Fig. 3.

Lamellocyte differentiation in ligated and infected-ligated Hemese-GFP larvae. (A) The lymph gland (arrow) stained with anti-Hemese (red) is located anterior to the ligature (arrowhead). (Scale bar: 100 μm.) Hemocytes isolated from the anterior and the posterior parts of (B and C) uninfected, and (D and E) infected larvae. (B–E) Lamellocytes are visualized by anti-L1 staining (red). GFP-expressing lamellocytes in the posterior part are shown by arrows. The numbers are the average numbers of all hemocytes and lamellocytes in the anterior and posterior parts. (Scale bar: 20 μm.) (F) The dots indicate the individual values. All, number of all hemocytes; Lam, number of lamellocytes. (G) Analysis of the encapsulation reaction in the ligated-infected larva. The wasp eggs dissected from the anterior part are not melanized (arrows). (Scale bar: 100 μm.)

Fig. 4.

Phenotype of the lymph gland and the circulating hemocytes in Hemese-GFP larvae after wasp infection; (A and D) 24 h, (B and E) 48 h, and (C and F) 72 h after wasp infection. (A–C) confocal Z stack images of the lymph glands, (D–F) circulating hemocytes from the same individuals as the dissected lymph glands above. The lamellocytes (arrows) were immunostained for the L1 antigen (red). An asterisk indicates one of the enlarged secondary lymph gland lobes. (Scale bars: 20 μm.)

Fig. 5.

Morphology of the lymph gland and phenotype of the circulating hemocytes of wasp-infected hml-GFP larvae; (A and D) 24 h, (B and E) 48 h, and (C and F) 72 h after wasp infection. (A–C) The posterior signaling centers (arrows) of the lymph glands are visualized with anti-collier staining (red), on confocal Z stack images. (D–F) Circulating hemocytes from the same larvae as the dissected lymph glands above are immunostained for the L1 antigen. Lamellocytes in circulation are marked with arrowheads. An asterisk indicates one of the enlarged secondary lymph gland lobes. (Scale bars: 20 μm.)

Fig. 6.

The lymph gland, and the circulating hemocytes of the hdc^B5 larva 72 h on parasitic wasp infection. The P{lacZ-un1}hdcB5 enhancer trap line was used as a lymph gland marker. (A and C) Confocal section of the lymph gland, (B and D) circulating hemocytes. (A and B) The headcase-LacZ expression is visualized by anti-β-galactosidase staining (green), the lamellocytes are immunostained for the L1 antigen (red). (C and D) The corresponding DIC or phase contrast and DAPI staining of the same fields. (Scale bars: 20 μm.)

Fig. 7.

Lamellocyte differentiation in transplanted Hemese-GFP-expressing posterior sessile hemocytes (4) dissected from the recipient Oregon-R wild-type strain. (A) Posterior part of a recipient Oregon-R larva. (B–D) Circulating hemocytes isolated from the recipient Oregon-R larva (B) GFP (green), (C) phase contrast and DAPI, (D) merged GFP and anti-L1 staining (red). Arrows indicate the transplanted sessile hemocytes; the arrowhead indicates the melanized site of injection. [Scale bars: A (100 μm) and B–D (20 μm).]