STAT [9,19], Toll [20] and JNK pathways [21]. Recently it has been suggested that the Friend of GATA protein U-Shaped (Ush) may be the target of these pathways during lamellocyte differentiation [22,23]. Significantly, these authors suggest that lamellocytes arise from plasmatocytes, indicating that a reassess- ment of lamellocyte origin is required. In the course of a misexpression screen to identify novel regulators of hemocyte development [14], we uncovered the transcriptional repressor CG11798 (also known as charlatan (chn) or taco). Chn was first isolated in a genetic screen for mutations affecting embryonic peripheral nervous system (PNS) development [24] and was subsequently shown to be an important regulator of achaete/scute gene expression [25]. Chn is a putative transcription factor that contains 5 zinc fingers and shares similarity with the human neuronal restrictive silencing factor/repressor element RE- 1 silencing transcription factor (NRSF/REST) [26]. Importantly, Chn interacts with the Drosophila homologue of the NRSF/REST co-repressor, CoREST [26]. CoREST forms the core of a transcription repressor complex that includes CtBP, HDAC-1/ HDAC-2 and LSD1, and exhibits histone deacetylase and histone demethylase activities [27–29]. Through its associated repressor complexes NRSF/REST binding is speculated to erase histone marks of active transcription (H3K4me2 and H3K9Ac), establish the repressive histone mark (H3K9me2), and repress transcription [27]. In this report we show that Chn over-expression is able to trigger lamellocyte differentiation. Interestingly, Chn over-expres- sion appears capable of transforming plasmatocytes into lamello- cytes. Our results suggest an alternative pathway for the origin of lamellocytes. Results Chn overexpression increases lamellocyte number During a screen to identify new regulators of hemocyte development [14] we uncovered the putative transcriptional repressor CG11798 (also known as Charlatan (Chn) or Taco [24–26]). To investigate further the effect of Chn on hemocyte development we overexpressed Chn in larval plasmatocytes and crystal cells using the Peroxidasin-GAL4 (Pxn-GAL4) driver. The Pxn- GAL4 driver line also contains a UAS-GFP reporter that enables plasmatocytes and crystal cells to be visualized live in the semitransparent larvae. Pxn-GFP expressing sessile plasmatocytes (Fig. 1A) and circulating plasmatocytes (Fig. 1B) could be detected in the Pxn-GAL4 driver control but were rarely observed in Pxn- GAL4 x UAS-chn larvae (Fig. 1E,F). Interestingly, loss of Pxn-GFP expression was not due to the absence of hemocytes as staining using the pan-hemocytic marker anti-Hemese [30] revealed that hemocytes were still present in Chn over-expressing larvae. Rather, hemocytes observed after Chn over-expression were larger than typical plasmatocytes (arrowheads, compare Fig. 1C,G), corresponding in size to lamellocytes. Crystal cells were not affected by Chn over-expression as melanized crystal cells were still detected in heat-treated Pxn-GAL4 x UAS-chn larvae (Fig. S1). Antibody staining using the lamellocyte marker MAb L1 [31] confirmed that Chn over-expression in plasmatocytes triggered lamellocyte differentiation (Fig. 1D,H), with lamellocyte frequency increasing from 0.5% in the Pxn-GAL4 driver control to 25.6% (Fig. 1I). Two classes of MAb L1 positive cells were detected. The first corresponded to mature lamellocytes with diameter .40 mm (closed arrowheads in Fig. 1H). The second consisted of cells of diameter between 10–40 mm (open arrowheads in Fig. 1H). These were sized between a typical plasmatocyte and mature lamello- Drosophila Lamellocyte Origin cytes and feasibly represent differentiation intermediates between plasmatocytes and mature lamellocytes. Pxn-GAL4 is also expressed in crystal cells and at low levels in the fat body [14]. By over-expressing Chn using either the crystal cell-specific driver lz-GAL4, or the fat body-specific driver Lsp2- GAL4 we could exclude these tissues as the source of lamellocytes. Neither lz-GAL4 nor Lsp2-GAL4 significantly increased lamellocyte frequency when used to over-express Chn (Fig. 1I, P value .0.05). In contrast, other plasmatocyte-expressed GAL4 drivers were able to induce lamellocyte differentiation when used to over-express Chn. For example, He-GAL4, which is expressed in about 30% of all hemocytes [14], increased lamellocyte frequencies to 20.5%, while Hml-GAL4, which is expressed in a sub-population of plasmatocytes [32], raised frequencies to 4.9% (Fig. 1I). In He- GAL4, UAS-GFP x UAS-chn or Hml-GAL4, UAS-GFP x UAS-chn larvae GAL4-mediated expression of GFP is sustained after Chn over-expression. Double staining of hemocytes isolated from these larvae identified cells that were both GFP-positive and MAb L1- positive (Fig. S2), showing that Chn-induced lamellocyte differen- tiation by these drivers is cell autonomous and that Hml-GAL4 expressing plasmatocytes can be transformed into lamellocytes. The lower numbers of lamellocytes observed after Chn over- expression using Hml-GAL4 compared with Pxn-GAL4 is most likely due to fewer hemocytes expressing Hml-GAL4 (35%) compared with Pxn-GAL4 (96%). When lamellocyte number after Chn over-expression is normalized to the number of hemocytes expressing each driver we observed comparable transdetermina- tion rates (Fig. S2). The ability of Chn over-expression to induce lamellocyte differentiation compared well with other transcription factors and known regulators of lamellocyte differentiation. As shown in Figure 1J, similar lamellocyte frequencies were observed after expression of dominant-negative U-shaped (Ush) variants [22] using Pxn-GAL4. However, the transcription factor Kruppel (Kr) and the co-activator Nejire (Nej) which had also been identified in our genetic screen to induce lamellocytes when expressed using Pxn-GAL4 [14], were much less efficient than Chn, with lamellocyte frequencies of 9.7% and 7.2% respectively observed (Fig. 1J). Significantly, overexpression of the early B cell factor homologue Knot (Kn) failed to induce lamellocyte differentiation, with lamellocyte frequency unchanged from the Pxn-GAL4 driver alone control (Fig. 1J). Kn was previously shown to be required for lamellocyte development although function is non-cell autono- mous [33], a conclusion supported by our results. As an additional control we observed that ectopic expression of another CoREST- interacting transcription factor, Tramtrack88 (Ttk88) [34], failed to induce lamellocyte development (Fig. 1J). Pulsed Chn expression increases lamellocyte numbers in circulation Pxn-GAL4 is first expressed in plasmatocytes during late embryonic stages [35] and persists during all larval stages [14]. In the previous experiments we examined lamellocyte differenti- ation at wandering third instar larval stages approximately 100 hrs after the onset of Chn expression in plasmatocytes. To exclude the possibility that lamellocyte induction by Chn was an indirect consequence of long-term expression of Chn we examined the effect of pulsed over-expression of Chn. Precise temporal control of Chn expression was overlaid on Pxn-GAL4 using the TARGET system [36]. In this approach, Pxn-GAL4 mediated induction is controlled by ubiquitous expression of a temperature sensitive variant of yeast GAL80 (GAL80ts). GAL80ts represses Pxn-GAL4 at 18uC, but is inactivated at 29uC, allowing onset of Pxn-GAL4 mediated expression to be controlled by temperature up-shift (see PLoS ONE | www.plosone.org 2 November 2010 | Volume 5 | Issue 11 | e14051