Fig. 5

Concurrent Expression of Syntaxin-18 SNARE Components Induces Bax-Dependent Apoptosis in a BNip1-BH3 Domain-Dependent Manner(A) Antiactivated caspase 3 antibody labeling of 7 hpf embryos injected with different RNA combinations of syntaxin-18 SNARE components: noninjection, syntaxin-18 SNARE mixture, syntaxin-18 SNARE mixture lacking BNip1b, syntaxin-18 SNARE mixture replaced with BNip1b (L114A), and syntaxin-18 SNARE mixture plus a mixture of MO-Bax1 and MO-Bax2. Cells with active caspase 3 were increased only in embryos injected with a mixture of mRNAs for all four syntaxin-18 SNARE components. (B) Top panels indicate 8 hpf embryos injected with the same RNA combinations shown in (A). The bottom panel shows a classification of the morphological defects of 8-hpf-injected embryos as embryonic cell death (class I), blown-up blastoderm during gastrulation (class II), nonuniform thickness of the blastoderm (class III), and normal morphology (class IV). (C) Percentages of 8 hpf embryos classified as classes I–IV in the injection experiments shown in (B) as well as the injection experiments for the syntaxin-18 SNARE mixture plus either MO-Bax1 or MO-Bax2. (D) Percentage of class I/II embryos. Green and black bars indicate the mean and SD, respectively. The numbers of injection experiments and t test p values are shown in Table S2 . ^*p < 0.05, ^**p < 0.01. (E) TUNEL staining of WT embryos expressing a mixture of all syntaxin-18 SNARE components and EGFP or only EGFP under the control of the atoh7 retinal enhancer. In embryos expressing a mixture of all syntaxin-18 SNARE components and EGFP, TUNEL-positive cells (magenta) are observed in the GFP-positive (green) retinal region, especially retinal ganglion cells (RGC; arrowheads), and anterior commissural (AC, arrowheads) and olfactory (olf, arrow) neurons. (F) The density of apoptotic cells (number/100 μm²) in retinas injected with DNA constructs encoding a mixture of syntaxin-18 SNARE components and EGFP or only EGFP. The number of examined retinas was n = 4 for each. Green and black bars indicate the mean and SD; ^**p < 0.01. Scale bars, 200 μm (A), 600 μm (B), and 50 μm (E). See also Figure S6.