|
Fig. 5
Knockdown of P54a and/or P54b RNA helicases in zebrafish embryos. (A?D) show the defects caused by MO-P54a, MO-P54b or MO-P54ab, which mostly affected posterior trunk structures and produce a bend at the end of the trunk (?tail curved?). (E) Morpholino efficacy was validated by reverse transcription-PCR (RT-PCR). Using specific primers for p54a or p54b mRNA in samples from embryos micro-injected with MO-ct or MO-P54ab. Primers for amplifying p54a and p54b, under normal conditions, produced bands of 241 and 242?bp, respectively. However, in samples from MO-P54b embryos, bands of 326 or 360?bp were observed due to an intron insertion. (F?I) ISH showing the expression of fgf8a and shha in WT and MO-P54ab 24?hpf embryos. (J,K) There were no obvious defects in embryos micro-injected with 15?ng of MO-ct or 300?pg of p54-mRNA. (L?Q) The ?tail curved? phenotype produced by MO-P54a, MO-P54b or MO-P54ab was rescued if the morpholino was co-injected with p54-mRNA. (R?W) With every micro-injection, a proportion of embryos developed normally (?tail normal?), but this proportion increased drastically upon rescue with p54-mRNA. (J?W) For each image, the percentage of embryos showing that phenotype is indicated, with the total number of embryos analyzed shown in parentheses. Control morpholino (MO-ct), p54a morpholino (MO-P54a), p54b morpholino (MO-P54b), mix of p54a and p54b morpholinos (MO-P54ab), ?in vitro? synthesized mRNA from p54a (p54-mRNA). ?in situ? hybridization (ISH), tail curved phenotype (TC), tail normal (TN).