Expression of rps19 in zebrafish embryos is specifically down-regulated by an antisense morpholino. (A) Zebrafish rps19 gene is composed of 6 exons. Antisense morpholinos corresponding to the sequence around the translational start site in exon 2 are designed to inhibit translation. Morpholinos derived from the intron 2 exon 3 boundary are designed to inhibit splicing. (B) Embryos start synthesizing rps19 mRNA at approximately 6 hpf; the low amount of rps19 mRNA at 0 to 3 hours postfertilization (hpf) is of maternal origin. RNA was pooled from at least 30 embryos at each time point. (C) Morpholinos targeting the splicing site of exon 3 of rps19 gene significantly decrease the level of rps19 mRNA message at 24 hpf: RT-PCR. in panel A show the positions of primers. Amounts of injected morpholino per embryo are indicated. Actin expression is used as a load control. A total of 60 to 100 embryos were injected with each dose; RNA was prepared from at least 30 embryos. A result representative of 3 experiments is shown. (D) Survival of zebrafish embryos depends on the dose of injected morpholinos. Complete blocking of RPS19 expression at 5 ng per embryo leads to death of all embryos at 48 hpf. The dose of 3.4 ng allows embryos to survive for approximately a week. Sixty to 100 embryos were injected with each dose, a representative experiment is shown. (E) Ribosomal biogenesis is defective in morphants. At 30 hpf, the amount of rRNA is lower in morphants. Total RNA was prepared from 30 wild-type or morphant embryos; the amount of loaded RNA is equivalent to 4 embryos.