Figure 5.

Asymmetric division of GaSCs. (A) Schematic diagram of the cell lineage marking system. After shifting the flies with genotype UAS-FLP/+; wg-Gal4 UAS-RFP/Act5C-FRT-y⁺-FRT-EGFP; tub-Gal80^ts/+ to 29°C, the Gal4 was activated and drove RFP (red) and FLP recombinase expression. The FLP recombinase then removed the ‘FLP-out’ cassette so that the constitutive actin5C promoter drove EGFP expression (green) permanently within all subsequent daughter cells. Flies with the genotype UAS-FLP/+; wg-Gal4 UAS-RFP/ Act5C-FRT-y⁺-FRT-EGFP; tub-Gal80^ts/+ (B–G, I, I′) were cultured at the permissive temperature (18°C, B) and then shifted to the restricted temperature (29°C, C–G, I, I′). At 18°C no EGFP/RFP expression (B). Flies shifted to 29°C for 12 hrs (C), 1 day (D, high magnification in D′), 2 days (E, E′, high magnification in E″, F, G). (G) 2 days (Z-stack merged). Division of GaSCs is highlighted in E′ (arrow) and E″ F (round dotted lines). In B–G the cardia was dissected and examined under confocal microscope without staining (live imaging). (H, H′) The flies with MARCM clones were dissected and stained with anti-GFP (green), anti-Ptc (red) and DAPI (blue). (I, and high magnification in I′) UAS-FLP/+; wg-Gal4 UAS-RFP/Act5C-FRT-y⁺-FRT-EGFP; tub-Gal80^ts/+ flies (2 days) were stained with RFP (red), GFP (green), Ptc (purple) and DAPI (blue). Ptc is expressed in GaSCs as highlighted by dotted lines in H, H′. Anterior is at the top in all panels except in I, I′, where anterior is in right. Scale bars: 10 µm (B–E); 2 µm (E′, F, G, D′ E″, I′); 5 µm (H, H′ I).