Drosophila Lamellocyte Origin Figure 2. Pulsed Chn overexpression induces lamellocyte differentiation. Pxn-GAL4, UAS-GFP; Ubi-GAL80ts x UAS-chn wandering stage third instar larvae were raised at 18uC (GAL4 repressed) and Chn was induced by up-shift to 29uC. (A–D) Lamellocytes are absent from circulation (A) 0 hr and (B) 12 hr after induction of Chn expression, but appear at (C) 18 hr, peaking at (D) 24 hr. (E) Proportion of lamellocytes ,40 mm (red line) or .40 mm (blue line) relative to total hemocyte number (marked by DAPI staining) Time-points are the mean +/2 s.d. of 10 determinations. (F) Proliferation index after Chn overexpression determined from number of anti-H3S10p antibody staining hemocytes relative to total hemocyte number (marked by DAPI staining) (G) Hemocytes freshly isolated from larvae grown at 18uC do not stain with MAb L1, but (H) after in vitro culture at 29uC for 24 hr to induce Chn expression many lamellocytes were detected, including mature (closed arrowheads) and intermediate (,40 mm) (open PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e14051