Fig. 4

The 24- and 7-nt insertions at the 3′ end of pou5f3 cause developmental retardation, increased Pou5f3 accumulation, and precocious 3′ end processing. (A) A schematic diagram of pou5f3 and the 3′ end sequences in the wild-type (WT) and pou5f324i/24i and pou5f37i/7i mutants. Sequences in blue and red denote an additional 3′ end and inserted nucleotides, respectively. Protospacer adjacent motif (PAM) and guide RNA (gRNA) sequences are highlighted in yellow and gray, respectively. (B) Lateral views of WT and pou5f324i/24i and pou5f37i/7i mutant embryos at 4, 6, and 8 hpf. Arrowheads indicate the marginal cells, showing the progression of epiboly. pou5f324i/24i and pou5f37i/7i mutant embryos exhibited delayed epiboly progression at 6 and 8 hpf. Similar results were obtained from three independent experiments. Scale bar, 200 μm. (C) Top: Immunoblotting of Pou5f3 in WT and pou5f324i/24i embryos at 0 and 3 hpf. Rpl11 is a loading control. Similar results were obtained from five independent experiments. (C) Bottom: Intensities of both Pou5f3 bands were quantified (means ± SD; n = 5). **P < 0.01 and *P < 0.05, Student’s t test. The intensities of the upper and lower Pou5f3 bands were quantified in fig. S5B. (D) PAT assay for zebrafish pou5f3 mRNA in WT and pou5f324i/24i embryos at 0 and 3 hpf. Similar results were obtained from two independent experiments. (E) Sequence analysis of the 3′ ends of pou5f3 mRNA in pou5f324i/24i embryos at 0 hpf. The lengths of 3′ ends (left) and poly(A) tails (right) are presented.