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Fig. 6 znf281b phenotype is rescued with exogenous znf281b-ALFA mRNA (A) Diagrams represent the synonymous nucleotide changes in the exogenous znf281b-ALFA mRNA and the experimental design for the rescue experiments. Exogenous znf281b-ALFA mRNA contains the 5? and 3' untranslated region (UTR) of ?-globin from Xenopus and not the endogenous UTRs. (B and C) RT-qPCR targeting znf281b CDS (B) or 3? UTR (C) for rescued experiment. Diagram for oligo pair positions is denoted on the right of each plot. Error bars represent SEM from three biological replicates, and significant differences were considered when p < 0.05 (Student?s t test). (D) Stacked bar plot represents proportions of developmental stages in each condition at 24 h post-fertilization. Representative images for each condition are below the plots, with quantification numbers under the pictures. See also Figure S7C 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. (E) Diagram for tail length quantification at 24 h post-fertilization and boxplot of tail lengths in ?m per condition. Data represent three biological replicates, and n denotes the number of embryos measured per condition. (F) Volcano plot shows differentially expressed genes between znf281b knockdown and pCas13d controls. Magenta highlights znf281b, and light blue dots highlight the first zygotic transcription wave observed from Baia Amaral et al.21 (G) Pie chart of down-regulated genes in a nanog knockout line from Lee et al.9 and shared down-regulated genes with this study?s znf281b knockdown (teal). n is the number of down-regulated genes in each section of the chart.