Fig. 2

PrP Morpholino Knockdown and RNA Rescue in Zebrafish Embryos

(A–F) Embryos were microinjected at one- to four-cell stages and photographed at 9 hpf. (A) Control embryos reach approximately 90% epiboly, as evidenced by the normal progression of the blastodermal margin (red arrowheads). (B) Epiboly in PrP-1 morphant embryos is severely impaired, and they remain arrested. (C) No appreciable morphological defects are seen in PrP-2 morphant embryos. In rescue experiments, the morpholino (MO) PrP-1 phenotype is reverted to different extents by microinjection of PrP-1 (D), PrP-2 (E), or mouse PrP (F) mRNAs. m, mouse; zf, zebrafish.

(G) Western blot (WB) analysis of 6-hpf embryo cell extracts shows that PrP-1 expression (<53-kDa band) is effectively suppressed by morpholino injection. Co, control.

(H and I) Embryos expressing the EGFP-PrP-1 fusion mRNA can overcome the MO PrP-1 arrest, normally reaching 75% epiboly at 8 hpf (H). In contrast, embryos expressing the corresponding control EGFP fusion mRNA remain arrested (I).

(J–M) At the prim-5 (24 hpf) stage, PrP-2 morphant embryos (K and M) show severe malformations in the head region, compared to control embryos (J and L). (J and K), lateral views; (L and M), dorsal views. e, eye; fb, forebrain; hb, hindbrain; mb, hindbrain.