Spätzle (Spz) cleavage is required for the Toll pathway activation in apoptosis-deficient flies.

(A) Schematic representation of the spz isoform variants and the mutation in the cleavage site. The cleavage site of wild-type spz, CGCGTT (Arg-Val) was mutated to the cleavage-resistant sequence CTTAAT (Leu-Asn) via CRISPR/Cas9. (B) Hatching rate of embryos from wild-type (w^iso31) or uncleavable Spz (spz^uc) female flies crossed with w^iso31 male flies. More than fifty embryos for each group. n = 3. (C) Representative images of embryos from wild-type (w^iso31) or spz^uc female flies 1–3 h or 6–8 h after egg laying. Scale bars: 100 μm. (D) Western blotting of the hemolymph samples of the female flies against Spz (anti-Spz C106 antibody). Genotypes are described in the figure. (E) Venn-diagram showing the number of upregulated genes in apoptosis-deficient (WP^QF>Dark^sh) male flies (Fold change > 1.5, versus WP^QF>+) and the number of downregulated genes in apoptosis-deficient flies with the spz^uc background (WP^QF>Dark^sh, spz^uc) (Fold change < 0.67, versus WP^QF> Dark^sh) with Benjamini–Hochberg adjusted P value < 0.05 after Wald test. (F) Quantitative RT-PCR of Drs in the whole body of the male flies 3 days after eclosion. n = 6. Genotypes are described in the figure. Data are mean with SEM, and each dot represents a replicate in B and F. In F, statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparison test. ns: P > 0.05; *: P < 0.05.