Drosophila Lamellocyte Origin arrowheads) forms. (I) Lamellocyte frequencies before (0 hr) and 24 hr after culture at 29uC. Data are the mean +/2 s.d. of 5 independent determinations. (J–L) Chn over-expression increases lamellocyte number in primary lymph glands (closed arrowheads), but (M–O) does not increase proliferation. Lamellocytes were not detected in the secondary lobes (open arrowheads) In (J–O) antibody staining is shown in green, DAPI-stained nuclei in purple. Scalebar in (A–H) denotes 50 mm, in (J–O) 200 mm. doi:10.1371/journal.pone.0014051.g002 Chn triggers lymph gland lamellocyte development We next tested if this feature is shared by lymph gland plasmatocytes. During larval stages the lymph gland is the site of a second wave of hematopoiesis, and generates hemocytes that are liberated at pupariation [12,13]. Lymph gland hemocytes have the ability to differentiate into lamellocytes in response to stimuli including, parasitic wasp infestation [9,12]. To determine whether Chn overexpression can also trigger lymph gland plasmatocytes to form lamellocytes we exploited the normal expression of Pxn-GAL4 in plasmatocytes and crystal cells in the cortical zone of the lymph gland [13]. As above, temporal control of Chn expression was overlaid over Pxn-GAL4 using the TARGET (Ubi-GAL80ts) system. MAb L1 staining revealed that Chn expression in lymph gland plasmatocytes induced the differentiation of large numbers of lamellocytes within 18 hr (Fig. 2K) and 30 hr (Fig. 2L) after up- shift to 29uC. Lamellocytes were detected in the cortical zone of the primary lymph gland lobes (closed arrowheads in Fig. 2K,L). These are the normal sites of plasmatocyte and crystal cell maturation and correspond to the domain of Pxn-GAL4 expression, indicating that the Chn-induced lamellocyte differentiation is cell autonomous. Importantly, lamellocytes were not observed in secondary lobes that contain undifferentiated prohemocytes (open arrowheads in Fig. 2K,L). Moreover, as noted for circulating hemocytes Chn induction did not increase proliferation of lymph gland hemocytes (Fig. 2M–O). In addition, the larval lymph glands remained intact, thus excluding the possibility of lamellocytes being released into circulation to account for increases in circulating lamellocyte numbers. Chn over-expression induces transcription of lamellocyte markers We next confirmed whether Chn over-expression induced expression of other lamellocyte markers. We purified hemocytes from Pxn-GAL4, UAS-GFP; Ubi-GAL80ts driver-alone and Pxn- GAL4, UAS-GFP; Ubi-GAL80ts x UAS-chn wandering third instar larvae that had been cultured at 29uC for 24 hours. mRNA was isolated and transcript abundance determined by real-time PCR. We confirmed that transcription of the 240 kDa subunit of Filamin (Filamin-240) is up-regulated 11 fold in hemocytes after Chn induction (Fig. 3A). Filamin-240 has previously been shown to be expressed exclusively on lamellocytes [37]. In a similar manner lamellocytes have previously been shown to stain with v antibodies against the b-integrins Myospheroid (Mys) and b - integrin (bInt-v) [19]. Up-regulation of the a-integrins a-PS4 and a-PS5 has also previously been shown to accompany ectopic lamellocyte differentiation observed in hopTum-l mutants [19]. We observed that Chn over-expression was accompanied by increased abundance of all these integrins, with Mys and bInt-v transcript levels increased 10 and 13 fold respectively, and a-PS4 and a-PS5 transcript levels increased 10 and 24 fold respectively (Fig. 3A). Thus Chn over-expression is accompanied by increased transcrip- tion of lamellocyte markers, confirming data obtained using MAb L1 antibodies. Conversely, Chn over-expression led to the repression of plasmatocyte markers. Consistent with results observed using the Pxn-GFP reporter, Chn over-expression reduced Pxn transcription by 35 fold 24 hours after Chn induction (Fig. 3B). Repression of Pxn transcription was accompanied by slight reduction in expression of Croquemort (Crq) and Hemolectin (Hml), while transcript levels of eater were essentially unchanged (Fig. 3B). These transcripts may not be direct targets of Chn and potentially require a longer period of Chn over-expression to observe significant reduction in expression levels. Endogenous Chn expression We next verified whether Chn is normally expressed in larval hemocytes. We compared the relative abundance of chn transcripts in purified hemocytes with abundance in whole third instar larvae and isolated fat body tissue. For comparison we assayed expression of hemocyte markers He and Hml and the fat-body marker Fat body protein 1 (Fbp1). As shown in Figure 4A, chn was expressed in wild- type hemocytes and enriched relative to whole third instar larvae 7.5 fold. This was comparable to the observed enrichment of the hemocyte markers He and Hml. In contrast chn expression in fat body was reduced relative to whole third instar larvae. To discriminate whether chn may be normally required for lamellocyte differentiation we examined the affect of RNAi knockdown of chn transcripts on wasp-induced lamellocyte differentiation. Control UAS-Dcr-2; Pxn-GAL4, UAS-GFP x w1118 and UAS-Dcr-2; Pxn- GAL4, UAS-GFP x UAS-chni 2nd instar larvae were infested with the parasitic wasp L. boulardi G486 for 12 hours and hemocytes isolated 30 hours later and lamellocytes visualized using MAb L1. As shown in Figure 4B, while lamellocyte frequency was almost 50% in control preparations, lamellocyte frequency in chn RNAi preparations was reduced to 25%, suggesting that knockdown of chn is partially able to block lamellocyte differentiation. In vitro differentiation of lamellocytes from plasmatocytes We next examined whether plasmatocytes can be induced to differentiate into lamellocytes in response to other signals. In particular, large numbers of lamellocytes have previously been observed in hopTum-l mutants, which encode a constitutively active variant of the Drosophila JAK [9,19]. We used these mutants to explore the transition from Pxn-GFP+ plasmatocyte to MAb L1+ lamellocyte. Antibody staining of hopTum-l; Pxn-GAL4, UAS-GFP hemolymph discriminated three hemocyte populations. As shown in Fig. 5A–D, GFP+/MAb L12 plasmatocytes (arrows), large (.40 mm) GFP2/MAb L1+ lamellocytes (closed arrowheads), and GFP+/MAb L1+ hemocytes (open arrowheads) were detected. Large (.40 mm) GFP+/MAb L1+ lamellocytes were never observed (Fig. 5D). The GFP+/MAb L1+ hemocytes (open arrowheads) detected were intermediate in size between plasma- tocytes (arrows) and mature lamellocytes (closed arrowheads), and resembled the ,40 mm MAb L1+ lamellocytes observed after pulsed over-expression of Chn. We postulate that these are double- positive intermediates between plasmatocytes and mature lamel- locytes. To provide support for this we purified GFP+/MAb L12 plasmatocytes and asked if these can differentiate into lamellocytes in vitro. The hopTum-l mutants provide a convenient system to test this, as the mutation is temperature-sensitive conditional. Thus, growth at 20uC weakly activates the JAK/STAT pathway and lamellocyte differentiation, while growth at 29uC induces strong JAK/STAT activation and lamellocyte differentiation. Hemocytes were prepared from hopTum-l; Pxn-GAL4, UAS-GFP larvae grown at PLoS ONE | www.plosone.org 5 November 2010 | Volume 5 | Issue 11 | e14051