Figure 3. Chn over-expression activates transcription of lamellocyte markers and represses transcription of plasmato- cyte markers. Pxn-GAL4, UAS-GFP; Ubi-GAL80ts x UAS-chn larvae were raised at 18uC (GAL4 repressed) and Chn was induced by up-shift to 29uC for 24 hours. Pxn-GAL4, UAS-GFP; Ubi-GAL80ts x UAS-chn larvae raised entirely at 18uC provided an uninduced control. mRNA was isolated from hemocytes from wandering stage third instar larvae and relative transcript abundance in Chn expressing relative to uninduced control hemocytes was determined by real-time PCR. RpL32 provided an endogenous control to normalize expression. Relative transcript abundance after Chn over-expression of (A) lamellocyte markers and (B) plasmatocyte markers. Data are mean and standard deviation of three independent determinations. doi:10.1371/journal.pone.0014051.g003 20uC. We confirmed that three hemocyte populations including GFP+/MAb L12 plasmatocytes (Fig. 5E, arrows) were present. Cells were sorted on a MoFlo cell sorter to select GFP+/MAb L12 Drosophila Lamellocyte Origin plasmatocytes (Fig. 5F, G). This purified plasmatocyte population was then cultured in vitro at 29uC to induce fully the JAK/STAT pathway. After 30 hours, both large (.40 mm) MAb L1+ lamellocytes (Fig. 5H, closed arrowheads), and smaller (,40 mm) MAb L1+ lamellocytes (Fig. 5H, open arrowheads) were detected. The lamellocyte frequency after 30 hours corresponded to 36.6% of cells (Fig. 5I), consistent with the differentiation of purified plasmatocytes into lamellocytes. Lineage tracing wasp-induced lamellocytes Lamellocyte differentiation induced by de-regulated activation of the JAK/STAT pathway or over-expression of Chn could be argued to be synthetic phenotypes. To confirm if lamellocytes differentiate from plasmatocytes in response to natural stimuli, we infested larvae with the parasitic wasp L. boulardi G486. Drosophila larvae were infested with female wasps for 12 hours, and circulating lamellocyte differentiation was followed 0, 24, 36 and 48 hours after infestation. We observed robust differentiation of circulating lamellocytes 36 and 48 hours after infestation (Fig. 6C, D). To determine whether wasp-induced lamellocytes derive from plasmatocytes we performed a lineage tracing experiment using the act5C.stop.lacZ flip-out system (Fig. 6E, [38]). We isolated circulating hemocytes from third instar larvae 36 hours after infestation and stained using MAb L1 and anti-lacZ antibodies. Pxn-GAL4-mediated FLP expression activated lacZ-expression in 31.3% of hemocytes (Fig. 6G). Although the frequency of lacZ+ cells was lower than expected, as Pxn-GAL4 is expressed in 95% of hemocytes [14], it was sufficient to trap lacZ+/MAb L1+ lamellocytes (Fig. 6F), including mature lamellocytes (closed arrowheads) and intermediates (closed arrows). Quantitation revealed that 26.4% of hemocytes were lacZ+/MAb L1+, of which 19.4% were intermediates (,40 mm), and 7.2% were the typical large lamellocyte form (Fig. 6G). lacZ2/MAb L1+ lamellocytes (open arrows) and lacZ+/MAb L12 hemocytes (open arrowheads) were also detected. Phagocytes can differentiate into lamellocytes Finally we examined whether plasmatocytes that differentiate into lamellocytes are na¨ıve, or whether plasmatocytes that have been conditioned by challenge may also differentiate into lamellocytes. To do this, we tested whether plasmatocytes that are active phagocytes can differentiate into lamellocytes. Pxn- GAL4, UAS-GFP; Ubi-GAL80ts x UAS-chn larvae were cultured at 18uC. Larvae were injected with fluorescent polystyrene micro- spheres and allowed to recover at 18uC for 2 hours. During the recovery period, the microspheres are actively engulfed by phagocytically active plasmatocytes, marking these plasmatocytes during subsequent analysis. After the recovery period, larvae were shifted to 29uC for 24 hrs to induce lamellocyte differentiation. Hemocytes were then isolated and stained using MAb L1 antibodies to detect lamellocytes and rhodamine phalloidin to reveal cell boundaries. As shown in Figure 7, confocal microscopy detected internalized beads in MAb L12 plasmatocytes (open arrows). Internalized beads could also be detected in MAb L1+ lamellocytes (closed arrows), suggesting that these originated from phagocytically active cells. Discussion Drosophila provide a genetically tractable model system to investigate cellular innate immune function [1–3]. In this report we have examined the origins of lamellocytes, which are Drosophila hemocytes that differentiate in response to parasite infestation. We show here that over-expression of the CoREST-interacting PLoS ONE | www.plosone.org 6 November 2010 | Volume 5 | Issue 11 | e14051