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Published on: February 19, 2016
Knockdown of two splice variants of Ca(2+)/calmodulin-dependent protein kinase Iδ causes developmental abnormalities
Yukako Senga1, Tadashi Nagamine, Isamu Kameshita
1Department of Life Sciences, Kagawa University, Kagawa, Japan.
Abstract:
We isolated cDNA clones for zebrafish Ca(2+)/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 36hpf but zCaMKIδ-S began to appear at 60hpf 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.
Insights
Two zebrafish Ca(2+)/calmodulin-dependent protein kinase I (zCaMKI) δ isoforms, zCaMKIδ-L and zCaMKIδ-S, exhibit distinct expression patterns. Their kinase activity is essential for normal zebrafish embryogenesis, as demonstrated by gene knockdown and rescue experiments.
Area of Science:
- Developmental Biology
- Molecular Biology
- Biochemistry
Background:
- Ca(2+)/calmodulin-dependent protein kinase I (CaMKI) is a crucial enzyme involved in various cellular processes.
- Understanding the specific roles of CaMKI isoforms in vertebrate development is essential.
Purpose of the Study:
- To isolate and characterize zebrafish Ca(2+)/calmodulin-dependent protein kinase I (zCaMKI) δ isoforms.
- To investigate the spatial and temporal expression patterns of zCaMKIδ isoforms.
- To determine the role of zCaMKIδ kinase activity in zebrafish embryogenesis.
Main Methods:
- cDNA library screening for zCaMKI δ isoforms.
- Recombinant protein expression and characterization in E. coli.
- Western blotting with isoform-specific antibodies for expression analysis.
- Morpholino-based gene knockdown and rescue experiments in zebrafish embryos.
Main Results:
- Two splice variants, zCaMKIδ-L and zCaMKIδ-S, were identified with distinct C-terminal sequences.
- Recombinant isoforms showed similar catalytic properties but different expression profiles.
- zCaMKIδ-L expression starts at 36 hpf, while zCaMKIδ-S appears at 60 hpf.
- zCaMKIδ-S is brain-predominant, zCaMKIδ-L is widely distributed, abundant in skeletal muscle.
- Gene knockdown resulted in severe embryonic morphological abnormalities (short trunk, kinked tail, small head).
- Phenotypes were rescued by wild-type zCaMKIδ but not by a kinase-dead mutant.
Conclusions:
- The kinase activity of zCaMKIδ is critical for zebrafish embryogenesis.
- Differential expression of zCaMKIδ isoforms suggests distinct functional roles during development.
