Fig. 1

Fig. 1

. s411 carry a mutation in the prkd2 gene. (A,B) Bright field image analysis of a wild-type zebrafish embryo compared to an s411 mutant embryo at 72 hpf. Heterozygous adults that carry the s411 recessive mutation give 25% offspring mutant embryos exhibiting heart edema, inadequate blood circulation leading to complete outflow tract stenosis and blood regurgitation by 72 hpf. Scale bars: 500 μm. (C) Bright-field image analysis prkd2-targeted morpholino-injected embryo at 72 hpf. MO-prkd2-injected embryos resemble the s411 phenotype possessing the same features as s411 mutants (three replicates, n>50). Scale bar: 200 μm. (D) Bulked segregant analysis and further genetic mapping positioned the s411 mutation on chromosome 15. Recombination analysis on 865 embryos, positioned the mutation initially between the markers, z6895 and z51478 and further fine-mapping positioned the mutation between markers we developed in the overlapping BACs: CU062627 and BX924136. (E) The s411 embryos carry an A to G mutation that translates to a Threonine (T) to Alanine (A) amino acid change. (F) Blast analysis showed that the threonine T757 (T714 in humans) is a conserved amino acid at a highly conserved region of the C-terminus PRKD2 kinase, between several species. (G) Schematic representation of zebrafish Prkd2 kinase. It consists of 923 amino acids and the main domains of the enzyme are indicating: two cysteine-rich motif domains (cys1 and cys2), a pleckstrin homology domain (PH), and the C-terminal catalytic domain where the PKC-phosphorylation sites, Ser749 and Ser753 reside. It is also highlighted the position of s411 mutation (A to G), a previously identified zebrafish mutation with a similar phenotype (Y849) and the position of premature stop codon after MO injection resulting to defective splicing.