Drosophila Lamellocyte Origin Figure 1. Chn over-expression increases lamellocyte number. Chn overexpression decreases (A,E) sessile (asterisk) and (B,F) circulating plasmatocyte numbers. (C, G) Anti-Hemese staining detects hemocytes after Chn expression although hemocyte size is increased (arrowheads) (D,H) MAb L1 staining confirms that Chn expression increases lamellocyte number. Circulating hemocytes were isolated from (B–D) Pxn-GAL4, UAS-GFP x w1118 and (F–H) Pxn-GAL4, UAS-GFP x UAS-chn third instar larvae. Antibody staining is shown in green, DAPI-stained nuclei in purple. Scalebars denote 50 mm. (I) Lamellocyte frequency after Chn expression driven by different GAL4 lines. Control driver cross with w1118 provides a reference. (J) Comparison of lamellocyte differentiation induced by expression of other transcription factors with Pxn-GAL4. Each data point in (I,J) is the mean +/2 s.d. of at least 5 assays. doi:10.1371/journal.pone.0014051.g001 Fig. S3). This provides a defined time point from which the time course of Chn-induced lamellocyte differentiation can be assayed. Larvae were cultured at 18uC until the third larval instar and then shifted to 29uC to induce Chn expression in plasmatocytes. Lamellocytes were rarely seen in larvae 0 hr (Fig. 2A), or 12 hr after up-shift to 29uC (Fig. 2B). However, lamellocyte number increased dramatically at 18 hr (Fig. 2C) and 24 hr (Fig. 2D) after up-shift. The lamellocyte frequency observed at 24 hr was 36.6%, equivalent to that observed after long-term expression of Chn (Fig. 1I). No significant changes in hemocyte proliferation (measured by anti-H3S10p staining) were observed at any point after up-shift to 29uC, with proliferation indexes consistently less than 1% (Fig. 2F), indicating that lamellocyte increase after Chn overexpression was not caused by over proliferation of a small pool of existing lamellocytes or precursors. Moreover the larval lymph glands remained intact over this time course (Figure 2K,L) suggesting that it is unlikely that lamellocytes released into circulation due to fragmentation of the lymph glands account for the observed increases in circulating lamellocyte numbers. As observed after long term Chn expression, two classes of MAb L1+ cells were detected i) mature lamellocytes .40 mm in size and, ii) lamellocytes ,40 mm in size, intermediate between plasmato- cytes and mature lamellocytes. However, lamellocytes larger than 40 mm showed a lag in appearance relative to those smaller than 40 mm (Fig. 2E). Thus at 24 hr after Chn induction, small MAb L1+ cells were 29% of hemocytes, while mature MAb L1+ lamellocytes only comprised 6.7%. This is consistent with lamellocytes originating from a small precursor that increases in size as lamellocytes mature. We next examined whether circulating hemocytes purified from Pxn-GAL4; GAL80ts x UAS-chn wandering third instar larvae could differentiate into lamellocytes in isolation in vitro. Circulating hemocytes were isolated from larvae that had been cultured at 18uC. Few of these were MAb L1+ lamellocytes (Fig. 2G). However, after culture in vitro at 29uC for 24 hours to initiate Chn over-expression, many MAb L1+ lamellocytes were detected (Fig. 2H). Quantitation of lamellocyte frequencies revealed that lamellocyte abundance increased from 1.3% to 40% after Chn induction in vitro (Fig. 2I). These data confirm that lamellocytes detected in circulation after Chn induction can arise from progenitors that exist in circulation prior to Chn over-expression. This is interesting in the light of recent reports that sessile hemocyte compartments have the ability to differentiate into lamellocytes [15]. Our data suggest that this is a common property of hemocytes of embryonic origin, whether in circulation or in sessile compartments. PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e14051