Morphological phenotype of live pak4Δ2/Δ4 mutants.

(A) wild type and F₃ mutant fish at 2 dpf. (B) wild type and F₃ mutant fish at 6 months. Fish were first photographed for documentation followed by fin biopsy genotyping for confirmation. (C) Whole mount in situ hybridization of wild type and mutant embryos at 7-somite for scl. Lateral (left) and dorsal (right) views are shown. Red and blue arrowheads indicate anterior and posterior blood domains, respectively. (D) Whole mount in situ hybridization of wild type and mutant F₃ embryos at 30 hpf for mpo. wt, wild type EK. In panels A, C and D, Δ2/Δ4 refers to the mixed population of homozygous mutant and trans-heterozygous mutant embryos. Panel B shows individuals of each defined genotype, all of which were normal and fertile.

Testing for possible alternative translational initiation downstream from the mutation site.

(A) Construction of the PAK4-GFP fusions. The 52-UTR and open reading frame of pak4 were cloned upstream of an eGFP cassette. The TALEN mutation site is indicated by the red arrow. (B) Expression of the fusions in zebrafish embryos. PAK4-eGFP RNAs were synthesized by in vitro transcription and injected into one-cell stage embryos. Green fluorescence was monitored at 24 hpf. Only the wild type construct exhibited fluorescence. (C) Western blot analysis of PAK4-eGFP RNA-injected embryos. Embryos at 24 hpf were dechorionated and used for total protein extraction and Western analysis with a GFP-specific antibody. eGFP RNA was injected into embryos as a positive control. UI, uninjected wild type EK. wt, wild type PAK4-eGFP. Δ2, PAK4Δ2-eGFP. Δ4, PAK4Δ4-eGFP. Ponceau Red staining of the Western blot membrane is shown as a loading control.

Testing the ability of mutant RNAs to rescue the MO knockdown phenotype.

(A) Whole mount in situ hybridization of wild type and mutant embryos at 30 hpf for mpo. (B) MZpak4 knockdown-rescue data. Percentages of embryos showing undetectable, reduced or normal staining as compared to control for mpo expression at 30 hpf in (A). UI, uninjected wild type EK. MO, 6 ng pak4 MO cocktail. MO+wt, 6 ng pak4 MO cocktail plus 800 pg wild type PAK4-eGFP RNA. MO+Δ2, 6 ng pak4 MO cocktail plus 800 pg PAK4Δ2-eGFP RNA. MO+Δ4, 6 ng pak4 MO cocktail plus 800 pg PAK4Δ4-eGFP RNA. Only the wild type pak4 RNA resulted in significant rescue of mpo expression.

Testing for stress contribution to the pak4 MO phenotype.

(A) Injection of standard control MO (6 ng) into wild type and pak4 mutant embryos. Live embryos at 3 dpf were anesthetized and photographed. Images shown are representatives of 92 out of 98 for wild type and 17 out of 17 for mutant. (B) Heat exposure of embryos. Wild type and F₃ mutants were exposed to 35°C from blastula stage to 24 hpf. Control samples (28.5°C) were incubated for 5 hours longer than the heated embryos in order to compensate for different developmental rates.