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

We isolated cDNA clones for zebrafish Ca2+/calmodulin-dependent protein kinase I (zCaMKI) delta 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 zCaMKI delta -L (long form) and zCaMKI delta -S (short form), respectively. Although recombinant zCaMKI delta -L and zCaMKI delta -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 zCaMKI delta -L clearly appeared from 36 hpf but zCaMKI delta -S began to appear at 60 hpf and thereafter. zCaMKI delta -S was predominantly expressed in brain, while zCaMKI delta -L was widely distributed in brain, eye, ovary and especially abundantly expressed in skeletal muscle. The gene knockdown of zCaMKI delta 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 zCaMKI delta , but not with the kinase-dead mutant. These results clearly indicate that the kinase activity of zCaMKI delta plays a crucial role in the early stages in the embryogenesis of zebrafish.