Fig. s9

Loss of zygotic mis/dhx6 function in dhx16^hi4049 homozygotes leads to caspase-dependent programmed cell death. (A,B) dhx16^hi4049/dhx16^hi4049 homozygote (top) and unaffected sibling (bottom) at day 3 p.f., shown under DIC optics (A) and fluorescence optics to detect acridine orange labeling (B). The darkening of the dhx16^hi4049/dhx16^hi4049 homozygotes (asterisk) correlates with an above-background number of acridine orange-labeled cells. (C–F) Groups of unsorted progeny from dhx16^hi4049/+ heterozygotes, treated with either carrier solvent (C,E) or the general caspase inhibitor Boc-D-FMK (D,F) and visualized with DIC optics (C,D) and to detect acridine orange labeling (E,F). In control-treated clutches, the expected fraction (1/4) of embryos shows signs of necrosis associated with increased acridine orange labeling (asterisks). Exposure to caspase inhibitor reduces both the penetrance and expressivity of the necrosis phenotype, and these effects correlate with decreased acridine orange labeling. At this stage of development, several regions show acridine orange labeling (sensitive to caspase inhibitor) in control, wild-type embryos, such as the anteriormost region of the brain ((ab), see also Furutani-Seiki et al., 1996) and hatching gland (hg) cells overlying the yolk cell. Fluorescence can also be observed in the developing cloaca (c), although this labeling appears non-specific as it can be observed in the presence of caspase inhibitor (F). Acridine orange labeling in the anteriormost region of the brain, observed in wild-type embryos at this stage, appears reduced in mutant embryos (B), possibly because these embryos do not undergo the normal developmental events that lead to apoptosis in wild-type embryos in this region.