Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster

Sessile hemocytes as a hematopoietic compartment in Drosophila melanogaster ´ Ro´bert Ma´rkusa,1, Barbara Laurinyecza,1, Eva Kurucza,1, Viktor Hontia, Izabella Bajusza, Botond Siposa, Ka´lma´n Somogyia, Jesper Kronhamnb, Dan Hultmarkb,2, and Istva´n Ando´a,2 aInstitute

of Genetics, Biological Research Center of the Hungarian Academy of Sciences, P.O. Box 521, H-6701, Szeged, Hungary; and bDepartment of Molecular Biology, Umeå University, S-901 87 Umeå, Sweden Edited by Kathryn V. Anderson, Sloan-Kettering Institute, New York, NY, and approved January 28, 2009 (received for review February 22, 2008)

cellular immunity 兩 lamellocytes 兩 parasitoid wasp 兩 plasmatocytes 兩 niche

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n both vertebrates and invertebrates the blood cells undergo differentiation in spatially separated organs, in a characteristic time sequence (1, 2). In Drosophila, at least 3 main classes of blood cells, or 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. Last, a class of large flat cells, the lamellocytes, appears when the larvae are infected by parasitoid wasps. These latter cells participate in the encapsulation of the parasites. In the Drosophila embryo, the head mesoderm contributes to generate both embryonic and larval hemocytes. Later, the lymph glands develop as major hematopoietic organs in the larva, derived from the lateral mesoderm (3). The larval hemocytes are distributed in 3 major compartments: the lymph gland, the circulating hemocytes, and a population of sessile cells attached to epithelial tissues. A majority of the sessile cells are found in a banded pattern under the larval epidermis, but many are also found attached to the imaginal discs. In the posterior end of the larva, groups of sessile hemocytes are concentrated in 2 denser organ-like clusters (4). The immune challenge caused by a parasitic wasp activates a vigorous cellular immune response and leads to the development of large specialized cells, the lamellocytes, which are involved in the encapsulation and killing of the parasites. The encapsulation reaction has a similar function as the formation of granuloma in vertebrates. It is generally assumed that lamellocytes differentiate in the lymph gland (5–10), because lamellocytes have been observed to accumulate there in parasitized larvae (6, 7). The lamellocytes are the most characteristic cells in the course of the immune response to parasites such as the parasitoid wasp, Leptopilina boulardi, although a few lamellocytes also develop spontaneously in unchallenged late third instar larvae. We observed that, after immune stimulation by L. boulardi, the subepidermal sessile cells are detached from the epidermis, and www.pnas.org兾cgi兾doi兾10.1073兾pnas.0801766106

lamellocytes appear in the circulation (11), whereas the lymph glands remain intact. This observation suggests that the sessile hemocytes and their release may be involved in the cellular immune response and perhaps also in lamellocyte development. Accordingly, we set out to perform a systematic analysis of the site of lamellocyte development after an immune challenge. We used cellular and molecular markers to analyze the cells in the main hemocyte and hematopoietic compartments, after an infection by the wasp, L. boulardi. We used 4 independent approaches to monitor the fates of the sessile cells and the cells in the lymph gland. First, we investigated the sessile compartment and determined the changes in number and in the immune phenotype of the blood cells. Second, we physically separated the sessile tissue and the lymph gland by the application of a ligature, and determined the morphological and immunological phenotypes of the hemocytes in the separated compartments. Third, we analyzed the morphology and the immunological phenotype of the lymph gland with special emphasis on the first lobes, which have been considered to be the main source of lamellocytes and of the circulating hemocytes after immune induction. Fourth, GFP-expressing cells from a cluster of subepithelial hemocytes, suggested to be a posterior hematopoietic tissue (PHT; see ref. 4), were transplanted, and the immunological phenotype of the circulating blood cells of the recipient individuals was determined. Results Changes in the Hemocyte Numbers and Phenotypes in the Sessile Tissue and in Circulation on Immune Induction. To determine the cell

types, we used antibodies against NimC1 (12) and L1 (4, 13), immunological markers for plasmatocytes and lamellocytes, respectively. To visualize sessile hemocytes, we used HemeseGAL4, UAS-GFP.nls (Hemese-GFP) larvae, which express GFP in the nuclei of a large majority (⬇80%) of the sessile and circulating hemocytes (11). Using these markers, we monitored the changes in the hemocyte subsets on parasitic wasp infection in the sessile compartment and in the circulation. Three days after immune induction, the banded pattern of the sessile compartment had disappeared (Fig. 1B), and lamellocytes appeared in the circulation. The total number of hemocytes in the sessile tissue was significantly greater in uninfected than in infected larvae (Fig. 2A; P ⬍ 0.001, 1-sided), whereas the number of circulating hemocytes was significantly lower in uninfected than in infected larvae (Fig. 2 A; P ⫽ 0.0495, 1-sided). The Author contributions: R.M. and I.A. designed research; R.M., B.L., E´.K., V.H., I.B., and J.K. performed research; E´.K. contributed new reagents/analytic tools; R.M., B.L., B.S., K.S., D.H., and I.A. analyzed data; and R.M., D.H., and I.A. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1R.M.,

B.L., and E´.K. contributed equally to this work.

2To

whom correspondence may be addressed. E-mail: [email protected] or dan.hultmark@ ucmp.umu.se.

This article contains supporting information online at www.pnas.org/cgi/content/full/ 0801766106/DCSupplemental.

PNAS 兩 March 24, 2009 兩 vol. 106 兩 no. 12 兩 4805– 4809

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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.

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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 ␮m) and C–F (20 ␮m).].

decrease in the total number of sessile hemocytes in the infected individuals was of similar magnitude as the increase in the circulating compartment (see figure legend in Fig. 2 A), suggesting that the sessile hemocytes could be the main source of the newly recruited circulating cells. Wasp infection did not affect the percentage of mitotic cells in the sessile compartment (0.68 vs. 0.69 at 24 h, and 4.87 vs. 4.83 at 72 h after infection; as analyzed by anti-phospho-histone H3 staining). In the uninfected animals, all circulating plasmatocytes carried the NimC1 antigen (12), whereas the sessile hemocytes were heterogeneous in respect to the NimC1 antigen. Approximately

Fig. 3. Lamellocyte differentiation in ligated and infected-ligated HemeseGFP larvae. (A) The lymph gland (arrow) stained with anti-Hemese (red) is located anterior to the ligature (arrowhead). (Scale bar: 100 ␮m.) 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 ␮m.) (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 ␮m.)

60% of the sessile GFP-positive cells were negative for NimC1 (Fig. 1C), and none of these cells carried the L1 antigen (Fig. 1E). These cells, lacking both the NimC1 and the L1 antigen, are operationally termed ‘‘double negatives.’’ After infection, the majority of both double negative and NimC1-positive hemocytes were lost from the subepidermal compartment (Figs. 1D and 2 B and C; P ⫽ 0.002165, 1-sided and P ⫽ 0.01082, 1-sided, respectively). A proportion of the NimC1-positive cells are also released from the surface of the imaginal discs [supporting information (SI) SI Text, Fig. S1, and Table S1], contributing to the increased number of circulating plasmatocytes. Also, L1positive lamellocytes appeared in circulation, as well as in the sessile tissue, after infection (Figs. 1F and 2D). The number of lamellocytes in circulation was significantly greater in the infected larvae (Fig. 2D, P ⬍ 0.001). Lamellocyte Formation After Physical Separation of the Sessile Hemocytes from the Lymph Gland by Ligation. To investigate further

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. 4806 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0801766106

the roles of the sessile hemocytes and the lymph glands in the origin of lamellocytes, we took advantage of the different anatomical localization of these tissues. The Hemese-GFP expressing sessile hemocytes appear sequentially in time in the posterior to anterior direction during larval development (Fig. S2). In the third instar (120-h old) larvae, the sessile tissue is fully developed, giving a banded pattern corresponding to the body segments. In the second larval instar (96-h old), the developing sessile compartment is located near the posterior end (Fig. S2a), and the lymph gland is located in the midanterior region (Fig. 3A). This fact allowed us to separate the lymph gland and the Ma´rkus et al.

sessile compartment physically with a ligature in the middle region of the larva (Fig. S2b). We applied such ligatures to second instar Hemese-GFP larvae after they had been wasp-infected. Uninfected ligated larvae served as controls. The banded pattern of the sessile tissue disappears similarly to the unligated larvae 12 h after ligation (Fig. 1). We separately dissected the anterior and the posterior ends of the larvae 48 h after ligation, and immunostained the circulating cells for the L1 (Fig. 3B–E) and the NimC1 antigen, respectively. Differentiation of a few lamellocytes was induced by the ligation procedure itself in the uninfected individuals (Fig. 3C), confirming that a mechanical injury of the larva is a sufficient trigger for lamellocyte development (14). In the infected and ligated individuals, vigorous lamellocyte differentiation was also seen, but lamellocytes were observed exclusively in the posterior body half of both the uninfected and the infected individuals (Fig. 3E). The immunostaining for an independent lamellocyte marker, L2 (4), confirmed that lamellocytes differentiate in the posterior end of the ligated larvae (Fig. S3b). Anterior to the ligation, the majority of the hemocytes were plasmatocytes (Fig. S3a), and the lymph gland was intact (Fig. 3A), indicating that lamellocytes were not released from the lymph gland, and that lamellocyte differentiation did not occur at this site. These observations were supported by the differential hemocyte counts (Fig. 3F). In the anterior part, the number of lamellocytes in the ligated and ligated-infected individuals was not significantly different (P ⫽ 0.8676, 2-sided). In the posterior part, the number of lamellocytes was significantly lower in the ligated than in the ligated-infected larvae (P ⬍ 0.001, 1-sided), indicating that lamellocyte formation occurred exclusively in the posterior part. Also, the number of lamellocytes was independent of the site of infection, as detected by the presence of a melanotic spot on the cuticle and by the presence of the wasp larvae during dissection. These findings lend support to the conclusion that the lamellocytes that appear in the circulation in the course of a cellular immune response arise from a tissue outside the lymph gland. To test whether these lamellocytes retain their function, second instar larvae were ligated and then infected, and the encapsulation of the wasp egg was monitored. The anterior and posterior parts were dissected separately 48 h later. Out of 30 dissected larvae, 6 had wasp eggs in the anterior part and none of these wasp eggs were melanized (Fig. 3G), whereas 11 larvae contained encapsulated and melanized wasp eggs in the posterior parts. Thus, the encapsulation and melanization reaction occurred only where lamellocytes were present.

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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 ␮m.)

anterior lymph gland lobes, and compared it with the time course of lamellocyte appearance in the hemolymph. Partially disintegrated anterior lobes could still be recognized by staining with an antibody to collier, which is characteristically expressed in the posterior signaling centers of these lobes (9). At early time points, the lymph glands were still intact in most of the larvae (Fig. 5 A and B); 24 h after infection they were intact in 13 larvae out of 13, and at 48 h, they were intact in 15 larvae out of 16. In contrast, L1-expressing lamellocytes were detected in circulation in 3 larvae out of 13 at 24 h, and in 15 larvae out of 16 at 48 h (Fig. 5 D and E). At 72 h after infection, the anterior lobes were

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of experiments to test the possible involvement of the lymph gland in the generation of lamellocytes. First, we compared the expression of the L1 antigen in the lymph gland and in the circulating cells. At 24 h after wasp infection, a majority of the larvae still showed no sign of lamellocyte differentiation in the lymph glands; they were L1-negative in 13 larvae out of 17 (Fig. 4A). At the same time, L1-expressing hemocytes were already present in circulation in 15 of these larvae (Fig. 4D). These cells were still rounded and relatively small, probably representing early stages of lamellocyte differentiation. Later, at 48 h after infection, a few lamellocytes were seen in the lymph glands of 5 larvae out of 10 (Fig. 4B), whereas in 8 larvae, an increasing number of L1-expressing hemocytes were detected in the circulation (Fig. 4E). Last, at 72 h, L1-expressing hemocytes were still absent in the lymph glands of 8 larvae out of 16 (Fig. 4C), whereas circulating fully differentiated lamellocytes were present in all of them (Fig. 4F). In a separate experiment, we followed the disintegration of the Ma´rkus et al.

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 ␮m.) PNAS 兩 March 24, 2009 兩 vol. 106 兩 no. 12 兩 4807

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Timing of Morphological Changes in the Lymph Glands and the Circulating Hemocytes on Immune Induction. We carried out 3 sets

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Fig. 6. The lymph gland, and the circulating hemocytes of the hdcB5 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-␤-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 ␮m.)

missing in 2/3 of the larvae (12 out of 17). In these larvae, only the collier-positive cells of the PSC were detected (Fig. 5C, arrow), whereas the secondary lobes had increased in size (asterisk). Again, lamellocytes were present in the circulation in all 17 larvae at this time point (Fig. 5F). Third, we used the enhancer-trap line hdcB5 (15) as a marker for the hemocytes that originate from the lymph gland. In hdcB5 larvae, ␤-galactosidase is expressed in cells of the lymph gland, but not in circulating or sessile hemocytes (Fig. S4). At 24 and 48 h after wasp infection, no ␤-galactosidase-positive cells were detected in circulation. At 72 h, when the first lobes are disrupted (Fig. 6 A and C), ⬇8% of the circulating L1-positive lamellocytes and 11% of the plasmatocytes showed ␤-galactosidase expression in the nuclei (Fig. 6 B and D). This expression was relatively weak in lamellocytes. The ␤-galactosidase-positive lamellocytes and plasmatocytes all expressed the pan-hemocyte marker Hemese (Fig. S5). From these experiments, we conclude that only a minor fraction of the lamellocytes and plasmatocytes in the infected larvae originate from the lymph gland, and that these lymph gland-derived cells enter circulation at a relatively late stage. Lamellocyte Formation from Transplanted Sessile Cells. Because all

of the above results suggested a possible sessile origin of the lamellocytes, we used a direct approach to follow the fate of the sessile hemocytes by transplanting GFP-expressing subepidermal hemocytes from Hemese-GFP larvae into nonfluorescent larvae. The transplanted cells were taken from the dense posterior cluster of sessile hemocytes that we have proposed to be a PHT (4). The transplanted cells were all round in morphology, and showed no L1 expression (4). This setup allowed us to monitor the fate of the sessile cells independently of the circulating cells and the lymph gland. Because a mechanical injury is a sufficient trigger for induction of lamellocyte development (14), no wasp infection was required in these experiments. In successfully transplanted larvae, GFP-expressing hemocytes were visible through the cuticle (Fig. 7A). These larvae were dissected 72 h after the transplantation, and the circulating hemocytes were immunostained for the L1 antigen to score for 4808 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0801766106

Fig. 7. Lamellocyte differentiation in transplanted Hemese-GFP-expressing posterior sessile hemocytes (4) dissected from the recipient Oregon-R wildtype 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 ␮m) and B–D (20 ␮m).]

lamellocyte differentiation. From 11 successful transplantations, we could retrieve a total of 53 GFP-positive hemocytes. Of these cells, 15 expressed the L1 antigen, and the majority of them showed the large flattened morphology characteristic for lamellocytes (Fig. 7 B–D). This experiment clearly shows that the transplanted cell population can differentiate into lamellocytes. Discussion Our results suggest that the sessile hemocytes serve as a major hematopoietic compartment in the larva, and that this compartment is actively involved at the onset of the immune response as a source of lamellocyte precursors in response to an immune challenge. We found no evidence for increased mitosis in the sessile hemocytes; therefore, cell division may not be involved in the terminal differentiation of lamellocytes from this tissue. The lymph gland has usually been regarded as the main source of lamellocytes, because lamellocyte differentiation is observed in the lymph gland after immune stimulation (5–10, 16). However, our results show that lamellocytes are already present in circulation at a time when the lymph glands are still intact. Also, we have acquired direct evidence that lamellocyte differentiation can take place in the posterior body cavity, even if it is physically separated from the lymph gland. We also found direct evidence that subepidermal cells from the proposed PHT can differentiate into lamellocytes. Lymph gland-derived lamellocytes appear at a relatively late stage, and only in small numbers, but they may contribute to the consolidation of the capsule. Our findings highlight the role of the sessile subepidermal population of hemocytes in the immune response of the Drosophila larva, and show that they constitute a new, immunologically active hematopoietic site. This site could be the hematopoietic niche for the head mesoderm-derived population of larval hemocytes described by Holz et al. (3), similar to the hematopoietic niche in the lymph glands for hemocytes from the thoracic mesoderm (10, 17). We suggest that this novel compartment holds lamellocyte precursor cells, which on immune induction enter circulation and differentiate into lamellocytes. Further investigation of the sessile hemocytes and identification of the molecules involved in the regulation of this process will help us understand the molecular steps of differentiation from stem cells to immunologically active specialized blood cells. Ma´rkus et al.

Materials and Methods Fly Stocks. Wild-type Oregon-R, and white1118 and transgenic flies were kept on a cornmeal-yeast diet. Hemese-GFP (⫽Hemese-GAL4/UAS-GFP.nls) and hml-GFP (⫽ hml⌬-GAL4, UAS-GFP) transgenic flies (11, 18) were used to visualize and count the hemocytes on live or fixed samples. In hdcB5 (⫽P{lacZun1}hdcB5) larvae (15), ␤-galactosidase was visualized by indirect immunofluorescence. Immune Induction. For infection with the parasitic wasp, 50 second instar larvae were placed together with 5 female L. boulardi wasps of strain G486 overnight at 18 °C. The blood cells were analyzed 24, 48, and 72 h after the wasps were presented to the larvae. In each group, 6 –12 animals were analyzed, and the experiments were repeated twice. Immunofluorescent Analysis, Hemocyte Imaging, and Counting. Larvae were bled, and hemolymph samples prepared as described previously (4, 13). Specimens for analysis of lymph glands and sessile hemocytes were prepared as follows: the larvae were opened with 2 forceps creating small, subsequent fissures along the longitudinal axis, flattened, and immobilized with insect needles (Austerlitz Insect Pins, Minutiens 0.15 mm). The fat body and the digestive tract were removed, whereas the lymph glands were left in position. Then, the specimens were fixed and transferred to microscope slides. The hemolymph samples and the larval specimens were fixed with 2% paraformaldehyde for 15 min in PBS, washed 3 times 5 min in PBS, and then blocked with 0.1% BSA in PBS, supplemented with 0.01% Triton-X 100. We used mouse monoclonal antibodies to assign hemocytes to blood cell subsets. The anti-L1 antibody is a mixture of 3 different anti-lamellocyte antibodies (L1a, L1b, and L1c; see ref. 4), whereas the NimC1 antibody is a mixture of 2 antibodies that react specifically with plasmatocytes (12). Parts of the experiments shown here have also been reproduced with a second molecular marker for lamellocytes, the L2 antigen (4), with similar results. The mouse anti-collier antibody was used to visualize the cells of the posterior signaling center of the lymph glands (9), at a dilution of 1:100. The hemocyte-specific mouse monoclonal antibodies were used as neat supernatants in combination with biotin-conjugated anti-mouse Ig (DAKO) at a dilution of 1:500, and Cy3-labeled streptavidin (Amersham) at a dilution of 1:3,000; or visualized by Alexa Fluor 568conjugated anti-mouse Ig (Molecular Probes) at a dilution of 1:500. The rabbit anti-␤-galactosidase antibody (Polysciences) at a dilution of 1:300 was used in combination with Alexa Fluor 488-conjugated anti-rabbit Ig (Molecular Probes) at a dilution of 1:800. All sessile cells of each individual Hemese-GFP larva were counted on the flattened cuticles by fluorescence microscopy. The number of circulating hemocytes was determined as described (4, 13). Mitotically active hemocytes were detected by anti-phospho-histone H3 staining, as

Isolation of the Lymph Gland from the Sessile Tissue by Ligation. Having presented the wasps to the Hemese-GFP Drosophila larvae overnight, the larvae were immediately ligated in the middle region with a human hair. The larvae were kept in a humid chamber at 25 °C. The anterior and the posterior parts of the larvae were dissected separately 48 h later. Encapsulation Test on Ligated and Infected Larvae. Thirty Hemese-GFP Drosophila larvae were ligated in the middle region, and then mounted on microscope slides with double-sided tape in a humid chamber at 25 °C, and 20 L. boulardi wasps were put on them for 1 hour. The anterior and the posterior parts were dissected 48 h later, and the melanized and nonmelanized parasitic wasp eggs were counted. Transplantation of the Sessile Hemocytes. Hemese-GFP Drosophila larvae were dissected in Drosophila Ringer’s solution, all internal organs were removed, and the epidermis were intensively washed; the remaining sessile hemocytes from the posterior part were collected with a glass capillary and injected into Oregon-R wild-type host. The procedure was done under a Leica MZ-10F epifluorescent stereomicroscope. Hosts were kept in humid chamber, on filter paper, at 25 °C for 2 h; after wound healing, they were transferred to cornmeal-yeast vials. We analyzed the hosts 72 h later with an epifluorescent stereomicroscope; then, hemocytes were collected and immunostained. Statistical Analysis of Data. The significance of the differences between the samples was assessed by using the permutation test (perm. test, with exact P values) from the exactRankTests R package (R version 2.4.0 Patched 200611-25 r39997) (19, 20). The sidedness of the alternative hypotheses is indicated in the results. The box-and-whiskers plots were generated by using R (19). ACKNOWLEDGMENTS. We thank Olga Kovalcsik and Szilvia Ta´pai for technical help. The hml-GFP flies were kindly provided by U. Banerjee (University of California, Los Angeles), and the collier antibody by Miche`le Crozatier (Centre National de la Recherche Scientifique, Toulouse, France). This work was supported by Hungarian National Science Foundation OTKA Grants T048720, NI60442, K68830, and NK 78024, and by grants from the Swedish Research Council, the Go¨ran Gustafsson Foundation for Scientific Research, and the Swedish Cancer Society. 13. Kurucz E´, et al. (2003) Hemese, a hemocyte-specific transmembrane protein, affects the cellular immune response in Drosophila. Proc Natl Acad Sci USA 100:2622–2627. 14. Ma´rkus R, Kurucz E´, Rus F, Ando´ I (2005) Sterile wounding is a minimal and sufficient trigger for a cellular immune response in Drosophila melanogaster. Immunol Lett 101:108 –111. 15. Weaver TA, White RA (1995) headcase, an imaginal specific gene required for adult morphogenesis in Drosophila melanogaster. Development 121:4149 – 4160. 16. Jung SH, Evans CJ, Uemura C, Banerjee U (2005) The Drosophila lymph gland as a developmental model of hematopoiesis. Development 132:2521–2533. 17. Mandal L, Martinez-Agosto JA, Evans CJ, Hartenstein V, Banerjee U (2007) A Hedgehog- and Antennapedia-dependent niche maintains Drosophila haematopoietic precursors. Nature 446:320 –324. 18. Sinenko SA, Mathey-Prevot B (2004) Increased expression of Drosophila tetraspanin, Tsp68C, suppresses the abnormal proliferation of ytr-deficient and Ras/Raf-activated hemocytes. Oncogene 23:9120 –9128. 19. R Development Core Team (2006) R: A language and environment for statistical computing (R Foundation for Statistical Computing, Vienna, Austria), ISBN 3-90005107-0, available at http://www.R-project.org. 20. Hothorn T, Hornik K (2006) exactRankTests: Exact Distributions for Rank and Permutation Tests. R package version 0.8-16.

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1. Hultmark D (1994) Insect immunology. Ancient relationships. Nature 367:116 –117. 2. Hoffmann JA, Reichhart J-M (2002) Drosophila innate immunity: An evolutionary perspective. Nat Immunol 3:121–126. 3. Holz A, Bossinger B, Strasser T, Janning W, Klapper R (2003) The two origins of hemocytes in Drosophila. Development 130:4955– 4962. 4. Kurucz E´, et al. (2007) Definition of hemocyte subsets by cell-type specific antigens. Acta Biol Hungarica 58:95–111. 5. Lanot R, Zachary D, Holder F, Meister M (2001) Postembryonic hematopoiesis in Drosophila. Dev Biol 230:243–257. 6. Evans CJ, Hartenstein V, Banerjee U (2003) Thicker than blood. Conserved mechanisms in Drosophila and vertebrate hematopoiesis. Dev Cell 5:673– 690. 7. Crozatier M, Meister M (2007) Drosophila haematopoiesis. Cell Microbiol 9:1117–1126. 8. Sorrentino RP, Carton Y, Govind S (2002) Cellular immune response to parasite infection in the Drosophila lymph gland is developmentally regulated. Dev Biol 243:65– 80. 9. Crozatier M, Ubeda JM, Vincent A, Meister M (2004) Cellular immune response to parasitization in Drosophila requires the EBF orthologue collier. PLoS Biol 8:E196. 10. Krzemien J, et al. (2007) Control of blood cell homeostasis in Drosophila larvae by the posterior signalling centre. Nature 446:325–328. 11. Zettervall C-J, et al. (2004) A directed screen for genes involved in Drosophila blood cell activation. Proc Natl Acad Sci USA 101:14192–14197. 12. Kurucz E´, et al. (2007) Nimrod, a putative phagocytosis receptor with EGF repeats in Drosophila plasmatocytes. Curr Biol 17:649 – 654.

described previously (12). Fluorescence microscopy and indirect immunofluorescence analysis were carried out with a Zeiss Axioskope 2 MOT epifluorescence microscope. Images were taken with a Zeiss Axiocam digital camera. The lymph glands were analyzed with the aid of an Olympus FV 1000 confocal microscope.

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PNAS 兩 March 24, 2009 兩 vol. 106 兩 no. 12 兩 4809