Fig. 5. Temporal analysis of hemocyte maturation. (A) Hemese (He, red) is expressed by all hemocytes in the primary lymph gland of the second instar larva. (B,C) Maturation marker expression in second instar lymph glands. (B) Expression of Pxn (red) and hml-gal4/UAS-GFP (green) commence at approximately the same time during development. Although most cells express both markers (yellow), some express only hml-gal4/UAS-GFP (arrowhead), and others express only Pxn (arrow). (C) Cg-gal4/UAS-GFP (green) is expressed in a small subset of cells expressing Pxn (red). (D) P1 antigen (red) is first expressed in the early third instar lymph gland. Fewer cells are seen expressing P1-antigen than Cg-gal4/UAS-GFP (green). (E) Schematic representation of the order of appearance of plasmatocyte maturation markers. (F-G) Maturing hemocytes, prohemocytes and pre-prohemocytes in the second instar lymph gland. (F,F') Maturing hemocytes are marked by Pxn expression (F', red). These cells either express low levels of dome-gal4/UAS-GFP (green, white arrowheads) or no dome-gal4/UAS-GFP (yellow arrowheads). Prohemocytes do not express Pxn, but are marked by dome-gal4/UAS-GFP (F,F', arrows). Finally, pre-prohemocytes are located medially, close to the dorsal vessel (DV) and are characterized by the absence of both dome-gal4/UAS-GFP and Pxn expression (asterisks, blue only). These pre-prohemocytes do express Hemese (G; red, arrows). (H-K') The second instar PSC (arrows) are marked by Ser-lacZ (H,J,K'; red). Expression of upd3-gal4/UAS-GFP (I,J, green; RG, ring gland) initiates in the PSC (I, arrows) and expands later to other cells of the lymph gland (J, green). dome-gal4/UAS-GFP (H, green) and Pvr (K,K'; green) are not expressed in the PSC. (L-O) Gal4-based cell-lineage tracing in the larval lymph gland. (L) Schematic diagram of the cell-lineage marking system. Test stock flies of the genotype UAS-FLP, actin5C-FRT-STOP-FRT-lacZ is crossed to various Gal4-expressing lines (enhancer-gal4, UAS-GFP). Gal4 activates GFP (green) and FLP recombinase expression, which then removes the `FLP-out' cassette such that the constitutive actin5C promoter then drives lacZ expression (red) permanently within all subsequent daughter cells. (M) Cortical zone cells are derived from dome-gal4-expressing cells. The dome-gal4 reporter causes cortical zone (CZ) cells in the third instar lymph gland to be permanently marked with ß-gal expression (red) despite the lack of dome-gal4 expression (as assessed by GFP) in this zone. (N) As a positive control, third instar lymph gland cells were found to be permanently marked by ß-gal expression (red) because of twist-gal4 activation in the embryonic mesoderm, from which the lymph gland is derived. No GFP expression is detectable, indicating that twist-gal4 is no longer expressed in the third larval instar. (O) As a negative control, we show that cells of the PSC do not contribute to cortical zone cells. Lineage tracing using Serrate-gal4 revealed that PSC cells remain few in number and do not give rise to cortical zone cells. The majority of the Serrate-expressing cells appear yellow because of simultaneous expression of GFP (green) and ß-gal (red). To-pro-3 (blue) marks nuclei. Scale bars: 10 µm in A-M,O; 100 µm in N.