We have successfully introduced antisense oligonucleotide MOs into the ear of the 24 hpf zebrafish embryo. Embryos that were raised after injection and electroporation were viable. If the embryos survived the electroporation, they grew at normal rates compared to embryos that had received no treatment, as well as embryos that had only been injected and embryos that had only been electroporated.

We observed the effects of mif and mif-like MOs after ear injection and electroporation through labeling with phalloidin and acetylated tubulin. Phalloidin staining of actin filaments in sensory hair cells of the posterior macula indicates that electroporation of mif and mif-like MO at the 24-hpf stage caused changes in hair cell and/or stereocilia numbers when compared to embryos that were only injected with the MOs (Figure 3). This decrease in hair cell numbers is consistent with the findings of Shen et al. (submitted) that MOs to mif and mif-like cause a reduction in the number of hair cells in the saccular macula. The decrease in hair cell numbers in embryos that were injected with MOs and electroporated demonstrate that electroporation may aid in moving the MOs across the cell membrane, thereby increasing the extent of the morphological effects when compared to embryos in which the otic vesicle was only injected. Changes in the morphology of the statoacoustic ganglion were observed through acetylated tubulin staining. The extent of innervation by the statoacoustic ganglion was affected, as embryos that were injected and electroporated show decreased branching of the neurites (Figure 5). There may also be decreased condensation of the ganglionic cells and/or decreased number of ganglion cells, because the acetylated tubulin staining is significantly decreased in embryos that were both injected and electroporated. Because mif has been shown to be critical for nervous system development (Suzuki et al., 2004), the changes seen after electroporation may be the result of increased transfection of the mif and mif-like MO solution into the neuroblast cells.

Electroporation of antisense oligo morpholinos directly into a developing tissue is a useful tool for controlling the expression of a gene during that tissue's development, both spatially and temporally, in the embryo. Electroporation of mif and mif-like MOs into the tissues of the inner ear resulted in abnormal morphology of both the posterior macula and the statoacoustic ganglion. There was a reduction in the number of sensory hair cells in the posterior macula in embryos that had been injected and electroporated in comparison with embryos that had received no treatment or with embryos that had either been only injected or only electroporated. There was also a decrease in the degree of innervation of the posterior sensory patch by the statoacoustic ganglion and the size of the SAG. Electroporation is a valuable method for transfecting macromolecules into specific tissues, with the benefit of observing the primary effects of that particular DNA, RNA, or MO in the desired tissue and discriminating these from secondary effects on other tissues, including the brain, neural crest and periotic mesenchyme on inner ear development (Barald and Kelley, 2004).