Fig. 5

Rapid protein-level changes before genome activation elicit genome activation factors (A) Volcano plot highlighting down- (dark blue) and up-regulated (magenta) proteins between 0 and 2 h post-fertilization. Previously published genes with validated ZGA functions are labeled in black, and one uncharacterized candidate is labeled in magenta. (B) Diagram of experimental design utilizing CRISPR-Cas13d to knock down candidate genes. Two independent gRNAs targeting znf281b were used. (C) RT-qPCR targeting znf281b transcript in knocked down embryos at 4 h post-fertilization. Error bars represent SEM from at least three biological replicates per condition. Statistical significance was considered when p < 0.05 (Student?s t test). (D and E) Stacked bar plot represents proportions of developmental stages in each condition at 6 (D) and 24 (E) h post-fertilization. Representative images for each condition are below the plots, with quantification numbers under the pictures. The ?deformed? category means that embryos could not be assigned to any known developmental stage when analyzed. See also Figure S7E for more examples of mild and severe phenotypes from 24 h post-fertilization embryos. Error bars represent SEM from three biological replicates, significant differences were considered when p < 0.05 (Fisher?s exact test), and n denotes the number of embryos per condition. Scale bars represent 600 ?m for all images. (F) In situ hybridization chain reaction (HCR) representative pictures of one control and one of the knockdowns for znf281b g1. Dashed line denotes the embryo perimeter. (G) Fluorescent quantification of HCRs in arbitrary fluorescent units. n = 4 and n = 10 for controls and znf281b knockdown, respectively.