Knockdown of two splice variants of Ca2+/calmodulin-dependent protein kinase Iδ causes developmental abnormalities in zebrafish, Danio rerio

We isolated cDNA clones for zebrafish Ca2+/calmodulin-dependent protein kinase I (zCaMKI) δ isoforms by expression screening using cDNA library from embryos at 72-h post-fertilization (hpf). There are two splice variants with different C-terminal sequences, comprising of 392 and 368 amino acids, and they are designated zCaMKIδ-L (long form) and zCaMKIδ-S (short form), respectively. Although recombinant zCaMKIδ-L and zCaMKIδ-S expressed in Escherichia coli showed essentially the same catalytic properties including substrate specificities, they showed different spatial and temporal expression. Western blotting analysis using the isoform-specific antibodies revealed that zCaMKIδ-L clearly appeared from 36 hpf but zCaMKIδ-S began to appear at 60 hpf and thereafter. zCaMKIδ-S was predominantly expressed in brain, while zCaMKIδ-L was widely distributed in brain, eye, ovary and especially abundantly expressed in skeletal muscle. The gene knockdown of zCaMKIδ using morpholino-based antisense oligonucleotides induced significant morphological abnormalities in zebrafish embryos. Severe phenotype of embryos exhibited short trunk, kinked tail and small heads. These phenotypes could be rescued by coinjection with the recombinant zCaMKIδ, but not with the kinase-dead mutant. These results clearly indicate that the kinase activity of zCaMKIδ plays a crucial role in the early stages in the embryogenesis of zebrafish.