Figure 1. Pak1 is required for normal development of zebrafish.

(A) Transcripts for pak1 were detected by whole mount in situ hybridization at the stages indicated. Hours post fertilization (hpf). (B) Expression of pak1 at various stages in development by RT-PCR. (C) RT-PCR analysis of pak1 transcripts in morphants compared to WT and mismatch MO injected embryos at 24 and 48 hpf. (D) Immunoblot analysis of Pak1 in morphants compared to WT at 24 and 48 hpf. (E) Representative images of the moderate (MO moderate) and severe (MO severe) pak1 knockdown phenotypes as compared to the wild-type (WT) and mismatch morpholino (MM) injected embryos at 24 hpf and 48 hpf. (F) Quantification of pak1 knockdown phenotype at 24 hpf. (G) Morphology of embryos injected with the indicated MOs plus mRNA for wild-type of kinase-dead human Pak1 (hPak1), wild-type zebrafish Pak1 (zPak1), zPak2a, or zPak2b. (H) Quantitation of phenotypes at 24 hpf. * p<0.0001. (G) In situ hybridization for sox10 and foxD3 at 16-18 somites (lateral and cranial views). Arrows point to cranial neural crest expression in lateral view. In situ hybridization for crestin at the 16-18 and the 20 somite stage. Note lack of migration of neural crest in cranial view (arrows) and down the tail in the lateral view. (H) Trunk images of pak1 morphants lacking melanophores at 2 dpf. (I) In situ hybridization for mitfa. (J) Quantification of melanophores at 2 dpf. (K) Quantification of melanophores migration in pak1 morphants compared to mismatch MO injected embryos. (L) Cartilage staining (Alcian Blue) at 6 dpf. (M) (upper panel) WT embryos were injected with either PID (Pak inhibitor) or PID L107F (inactive Pak inhibitor) mRNA. Embryos are shown at 48 hpf; (lower panel) WT embryos were injected with expression vectors encoding PID or PID L107F driven by the sox10 promoter or the cmlc promoter, as indicated. Images were taken at 48 hpf.