Fig. 3.

Effects of JH signaling deprivation on the transcriptional profiles in embryonic legs of T. domestica. Volcano plots (A, C, E, G) and KEGG enrichment bubble maps (B, D, F, H; top 15 downregulated pathways) are generated from comparative analysis of embryonic leg transcriptomes in T. domestica. The embryonic legs were sampled at ED10 and ED11 (mixed in a ratio of 6:4) from control, KO-JHAMT, KO-CYP15A1, KO-Met, and KO-Kr-h1 embryos of G0 for RNA-seq. Differential expression analyses were conducted according to the schemes of KO-JHAMT vs. control (A, B), KO-CYP15A1 vs. control (C, D), KO-Met vs. control (E, F), and KO-Kr-h1 vs. control (G, H), respectively; genes with an adjusted P value < 0.05 (with no threshold for fold change) are designated as differentially expressed. Each group contained three independent biological replicates, and at least 200 embryos classified as class II or class I were pooled to generate one biological replicate. CRISPR/Cas9-mediated knockout of JH biosynthesis genes (JHAMT, CYP15A1) or JH receptor genes (Met) significantly suppressed JH signaling, as indicated by the expression level of Kr-h1 on the volcano plots. Oxidative phosphorylation, TCA cycle, and muscle contraction pathways are highlighted in blue on the bubble maps. I Venn diagram analysis of commonly downregulated, top 20 (fold change) annotated genes from the four groups of comparative transcriptomes. Genes that simultaneously appeared within at least three groups are listed, with two muscle activity-related genes, sarcoplasmic calcium-binding protein 1 and PDZ and LIM domain protein Zasp, highlighted in blue. Histological changes of the leg muscles from control (J), KO-JHAMT (K), and KO-Kr-h1 (L) embryos at ED11, as observed through transmission electron microscopy (TEM). Detailed magnifications of muscle fibers in J–L (red boxes) are shown in J’–L’. The Z-disks, muscle fibers, and mitochondria were indicated with the red, blue, and green arrows, respectively