Drosophila Lamellocyte Origin Figure 6. Lineage tracing reveals that wasp-induced lamellocytes arise from plasmatocytes. (A–D) MAb L1-staining (green) of hemocytes isolated from third instar larvae (A) 0 hr, (B) 24 hr, (C) 36 hr, and (D) 48 hr after 12 hr infestation with the parasitic wasp L. boulardi. DAPI-stained nuclei are shown in purple. Scalebar denotes 50 mm. (E) Schematic of the lineage tracing experiment. Pxn-GAL4 drives expression of FLP in plasmatocytes. FLP excises an FRT flanked transcription stop cassette from the act5C.stop.lacZ transgene, turning on expression of lacZ in a plasmatocytes. act5C-lacZ expression is then independent of Pxn-GAL4, marking cells that were plasmatocytes during subsequent lamellocyte differentiation. (F) MAb L1 (green) and anti-lacZ (red) antibody staining reveals lacZ+/MAb L1+ lamellocytes confirming the plasmatocyte origin of lamellocytes. Three classes of lamellocytes occur, lacZ2 lamellocytes (open arrows), and lacZ+ lamellocytes that are ,40 mm (closed arrows) or .40 mm (closed arrowheads) lacZ+/MAb L12 hemocytes indicated by open arrowheads. (G) 31.3% of hemocytes are lacZ+, of which 26.4% are lacZ+/ MAb L1+. Consistent with the origin of lamellocytes from small precursors, lacZ+/MAb L1+ cells ,40 mm in diameter and cells .40 mm in diameter are detected. doi:10.1371/journal.pone.0014051.g006 transcription factor Chn in plasmatocytes induces lamellocyte differentiation, both in circulation and in lymph glands. Our data indicate that Chn over-expression transforms plasmatocytes into lamellocytes. Consistent with this, we are able to detect double- positive intermediates between plasmatocytes and lamellocytes, and show that isolated plasmatocytes in vitro can be triggered to differentiate into lamellocytes following Chn over-expression. This property is not limited to Chn as we observed that other stimuli, including activation of the JAK/STAT pathway and the natural response to parasitic wasp infestation, also induced lamellocyte formation from plasmatocytes. Our data suggest that Chn may control lamellocyte development. Previously defined regulators of lamellocyte development include the transcription factor STAT92E, the FOG-1 homologue Ush, and the NURF chromatin remodelling complex. STAT92E functions as an inducer of lamellocyte development, as gain-of-function hopTum-l mutants that activate the JAK/STAT pathway cause lamellocyte over-production. In contrast, both loss-of-function ush and Nurf mutants exhibit increased lamellocyte numbers [19,22]. Like the homologous FOG-1-GATA-1 pairing, Ush modulates activity of the Drosophila GATA factor Srp [39] to favour plasmatocyte differentiation [22]. Recent data in mammalian systems indicates that FOG-1 mediates its effect on GATA-1 in part via recruitment of the transcriptional co-repressor NURD [40], suggesting that Ush functions similarly to repress expression of gene targets required for lamellocyte differentiation in plasmatocytes. Likewise, NURF also PLoS ONE | www.plosone.org 8 November 2010 | Volume 5 | Issue 11 | e14051