Kinesin-12(kif15) Expression And Functional Analysis During Zebrafish Neuronal Development

Objective Analyzing the temporal-spatial gene expression patterns of Kinesin-12 during the zebrafish embryonic development and the role of Kinesin-12 on neural development.Methods Conservational analysis of Kinesin-12 during vertebrate’s evolution using bioinformatics tools. Phylogenetic analysis of Kinesin-12 from several selected vertebrates. Investigating the expression of Kinesin-12 mRNA in early zebrafish development by whole mount in situ hybridization. Microinjecting Kinesin-12 mRNA or Morpholinos into Tg(hu C:egfp) zebrafish embryos and observing its effect on the neuronal morphology and the velocity of neuronal growth by Confocal laser scanning microscope and time-lapse imaging.Results The alignment of amino acid residue sequences and phylogenetic analysis indicate Kinesin-12 is highly conserved during vertebrate’s evolution.The whole mount in situ hybridization signals were detected at 2 cell, 1K cell, 50% epiboly and 75% epiboly stage. From 14 hpf to 30 hpf, the expression of Kinesin-12 was distributed to CNS, and was also shown in the otic-vesicle, retina, fin and peripheral olfactory organ. At 60 hpf, the expression of Kinesin-12 was concentrated in brain, but the hybridization signal was not detectable in the spinal cord at this stage. At 4 dpf and 5 dpf, the expression level of Kinesin-12 was strongly reduced.When expression of Kinesin-12 was overexpressed by injecting Kinesin-12 mRNA into Tg(huC:egfp) zebrafish embryos, we observed notably shorter axons and more branches compared to the control. By contrast, the axons were longer and less branches when Kinesin-12 was knockdown by injecting Kinesin-12 Morpholinos.Conclusions Kinesin-12 was expressed at cleavage stage, blastula stage and gastrula stage during the zebrafish embryonic development. The expression of Kinesin-12 was concentrated in CNS, otic-vesicle, retina and peripheral olfactory organ. Its overexpression or knockdown affected neuronal morphology and velocity of axonal growth, which indicates that Kinesin-12 plays an important role on the normal neural development of zebrafish.