Fig. 2. dark is essential for programmed and unprogrammed apoptosis. (A-D) Maternal and zygotic sources of dark were removed using a Dominant Female Sterile strategy (see Materials and methods). The resulting embryos lacked nearly all PCD, shown here by Acridine Orange (AO) staining (green). A and B show mid-staged embryos eliminated for maternal dark but heterozygous for zygotic dark; C and D show comparably staged embryos lacking both maternal and zygotic dark. Note that without a source of dark, embryos are head involution defective with only few AO-positive cells (C,D). (E-G) Requirement for dark in models of stress-induced cell death. Hemocyte aspirates from dark⁸² and wild-type (wt) wandering third instar larvae were treated with chemical stressors ex vivo and stained with CellTracker (see Materials and methods). Induction of apoptosis in wild-type (E) but not dark⁸² (F) hemocytes is exemplified here with micrographs taken 6 hours after Cycloheximide (CHX) treatment. (G) Quantification of apoptosis 6 hours after challenge with either CHX or a Smac mimetic (Li et al., 2004) are plotted as the incidence of cell death in percentages. Error bars indicate s.d.