Figure 7. Active phagocytes can differentiate into lamellocytes. Pxn-GAL4, UAS-GFP; Ubi-GAL80ts x UAS-chn larvae were raised at 18uC (GAL4 repressed) and injected with fluorescent polystyrene micro- spheres. Chn expression was induced by up-shift to 29uC for 24 hours to trigger lamellocyte differentiation. Isolated hemocytes were stained using MAb L1 antibodies to identify lamellocytes, and using rhodamine- phalloidin to distinguish cell boundaries. Engulfed microspheres were identified by intense yellow green fluorescence (which bleeds through into the red channel). A confocal Z stack was compiled through a MAb L12 plasmatocyte and a MAb L1+ lamellocyte. Projections along the X- and Y-axis confirmed that both contained engulfed microspheres. In panels rhodamine-phalloidin staining is shown in red, fluorescent polystyrene microspheres as yellow spots (open arrowheads) and MAb L1 staining is pseudocolored green. MAb L1+ lamellocyte is indicated by a closed arrow and MAb L12 plasmatocyte is indicated by an open arrow. doi:10.1371/journal.pone.0014051.g007 inhibits lamellocyte differentiation, in this case by preventing activation of targets of the JAK/STAT pathway [19]. The current biochemical data suggest that Chn is a transcrip- tion repressor as Chn recruits the co-repressor complex CoREST [26]. Indeed Tsuda and colleagues have shown that Chn over- expression represses Delta expression in the eye imaginal disk, while we have shown that Chn over-expression is accompanied by repression of some plasmatocyte markers. However, we have also shown that Chn over-expression leads to elevated expression of lamellocyte markers, and Escudero and colleagues [25] have demonstrated that Chn over-expression increases expression of the proneural genes Achaete and Scute. Our data do not allow us to discriminate whether Chn functions entirely as a transcriptional repressor or whether it may also activate transcription. However, the temporally-controlled Chn induction system (Pxn-Gal4 TAR- GET) that we have utilized here will allow the primary gene targets of Chn to be determined. By analyzing transcriptional profiles of hemocytes at defined time points after Chn over- expression the primary responders to Chn over-expression will be able to be identified. It will be possible to discriminate whether these targets are preferentially activated or repressed, and also subsequently determine recruitment of transcription co-activator or co-repressor complexes such as CoREST at these targets using chromatin immunoprecipitation (ChIP). Drosophila Lamellocyte Origin Our data demonstrating that lamellocytes can originate from plasmatocytes sheds new light on hemocyte lineages. As shown in Figure 8, current models of hemocyte lineages speculate that plasmatocytes, crystal cells and lamellocytes are distinct lineages that arise separately from a common stem cell or prohemocyte [1–3,12]. We propose, however, that prohemocytes generate either crystal cells or plasmatocytes. We suggest that plasmatocytes are a plastic population that can generate other frequently observed hemocyte types including lamellocytes. This model is strikingly reminiscent of the initial hemocyte lineages first proposed by Rizki in 1957 [41]. According to Rizki, prohemocytes were predicted to generate either crystal cells or plasmatocytes, with plasmatocytes differentiating further into activated cells (podocytes) and then lamellocytes [41]. This model has support from a number of experimental studies including this manuscript. Foremost amongst these are recent studies of hemocyte functions of Ush. Dominant-negative Ush variants are able to induce lamellocyte differentiation and it has been suggested that Ush regulates lamellocyte differentiation from a potential plasmatocyte [22]. Secondly, lamellocyte differentiation in response to Salmonella infection is blocked in decapentaplegic mutants with a corresponding increase in plasmatocyte number, suggesting that lamellocytes arise from plasmatocytes or a common precursor [23]. Two recent studies also suggest that plasmatocytes are a plastic population that may be able to differentiate into lamellocytes. Marking of embryonic plasmatocytes using the gcm-GAL4 or sn- GAL4 drivers and an act5C.stop.GAL4 flip-out transgene shows that lamellocytes that arise in larvae after wasp infestation may originate from cells that had expressed gcm-GAL4 or sn-GAL4 in embryos [42]. Similar results have also been observed using the act5C.stop.GAL4 flip-out transgene and Pxn-GAL4 and eater-GAL4 [43]. In both these cases the elicitor of the FLP/FRT activation event and the subsequent sustained marker are the same, namely GAL4 expression. However, in our lineage tracing experiments, GAL4 expression initiates the FLP/FRT activation of a distinct marker, lacZ protein. These data, taken together with our lineage tracing experiments and in vitro differentiation studies suggest that the plasmatocyte is an inherently plastic cell type that is capable of being reprogrammed to tailor immune responses to suit the infectious threats faced by the host. In humans, lymphocyte and leukocyte plasticity [44–46] has a significant impact on immune responses. An important future challenge is to establish the full spectrum of Drosophila plasmatocyte heterogeneity and exploit the utility of the Drosophila genetic system to dissect the mechanisms that regulate such leukocyte plasticity. Materials and Methods Fly strains and genetic crosses The following GAL4 drivers were used: w1118; Pxn-GAL4, UAS- GFP [35]; Hml-GAL4 [32]; lz-GAL4 [42]; Lsp2-GAL4; He-GAL4 [30]. UAS lines used were: UAS-chn [25]; UAS-ush-F13 (aa 302- 1191) [22]; UAS-kn [17]; UAS-nej [45]; UAS-ttk88 [34] and UAS-Kr [46]. For crosses, 5–10 driver line virgin females were mated with 5 males of each UAS strain. Progeny larvae were staged and analyzed as described in [14] and processed for confocal immunofluorescence microscopy as described below. Temporal control of GAL4-mediated Chn expression was using the TARGET system [36]. The w1118; Pxn-GAL4, UAS-GFP; GAL80ts driver was generated by crossing w1118; Pxn-GAL4, UAS- GFP; +/+ with w1118; +/+; GAL80ts (w; P{tubP-GAL80ts}2). Virgin driver females were crossed with UAS-chn males and crosses maintained at 18uC to repress GAL4. Progeny were shifted to PLoS ONE | www.plosone.org 9 November 2010 | Volume 5 | Issue 11 | e14051