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. 2013 Nov 26:2013:324839.
doi: 10.1155/2013/324839. eCollection 2013.

Expression of a Mutant kcnj2 Gene Transcript in Zebrafish

Ivone U S Leong  1 , Jonathan R Skinner  2 , Andrew N Shelling  3 , Donald R Love  4
Affiliations

Expression of a Mutant kcnj2 Gene Transcript in Zebrafish

Ivone U S Leong et al. ISRN Mol Biol. .

Abstract

Long QT 7 syndrome (LQT7, also known as Andersen-Tawil syndrome) is a rare autosomal-dominant disorder that causes cardiac arrhythmias, periodic paralysis, and dysmorphic features. Mutations in the human KCNJ2 gene, which encodes for the subunit of the potassium inwardly-rectifying channel (IK1), have been associated with the disorder. The majority of mutations are considered to be dominant-negative as mutant proteins interact to limit the function of wild type KCNJ2 proteins. Several LQT7 syndrome mouse models have been created that vary in the physiological similarity to the human disease. To complement the LQT7 mouse models, we investigated the usefulness of the zebrafish as an alternative model via a transient approach. Initial bioinformatic analysis identified the zebrafish orthologue of the human KCNJ2 gene, together with a spatial expression profile that was similar to that of human. The expression of a kcnj2-12 transcript carrying an in-frame deletion of critical amino acids identified in human studies resulted in embryos that exhibited defects in muscle development, thereby affecting movement, a decrease in jaw size, pupil-pupil distance, and signs of scoliosis. These defects correspond to some phenotypes expressed by human LQT7 patients.

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Figures

Figure 1
Figure 1
Average relative temporal and spatial gene expression and whole mount in situ hybridisation staining for kcnj2-12. (a) Average spatial gene expression profile. (b) Average temporal expression profile during embryonic development. Results for (a) and (b) were averaged across three experiments. The relative mRNA expression is quantified by qRT-PCR and all mRNA expression is relative to housekeeper genes ef1α and rpl13α. The error bars represent standard errors of the means. The tissues for each spatial gene expression profile were pooled from ten fish and 20–25 embryos were pooled for each time point of the temporal expression profile. (c) Left: side view of 24 hpf embryo showing staining throughout the whole of the body with the most staining in the brain, eyes, and somites (40x magnification; the black arrowhead indicated somite staining). Middle: a magnified side view of the defined staining in the somites (100x magnification). Right top: a magnified frontal view of the eyes and neurotube (100x magnification). Left bottom: dorsal view of staining of the otic vesicles and the somites (100x magnification; indicated by the white arrowheads). (d) Left: (top) dorsal view and (bottom) side view of 48 hpf stained embryo. Staining is strong in the brain, eyes, and slightly in the heart (40x magnification; heart staining indicated by a pink arrowhead and faint staining in the trunk indicated by a black arrowhead). Right: magnified view of the heart (100x magnification).
Figure 2
Figure 2
The average heart rate of embryos and the percentage of embryos unable to perform a full coil following the expression of Δ95–98 kcnj2-12, WT kcnj2-12, and combined Δ95–98 and WT kcnj2-12 proteins. (a) and (b) The average heart rate at 24 hpf and 48 hpf, respectively. (c) and (d) Representative images of embryos performing a full coil (c) and those unable to perform a full coil (d). (e) The average percentage of embryos unable to perform a full coil at 26 hpf. All results were averaged over three experiments and the error bars represent standard error for the means. The P values were calculated using one-way ANOVA. * P < 0.05, ** P < 0.01, and *** P < 0.001. For each experiment, each group contained ~60–70 embryos.
Figure 3
Figure 3
Gross morphology of somites of 26 hpf embryos expressing Δ95–98 kcnj2-12, WT kcnj2-12, and combined Δ95–98 and WT kcnj2-12 proteins under differential interference phase contrast (DIC) microscopy. (a) and (b) The somites of empty plasmid and uninjected control embryos. (c), (e) and (g) Embryos over-expressing different kcnj2-12 proteins that were able to perform a full coil. (d), (f) and (h) Embryos over-expressing different kcnj2-12 proteins that were not able to perform a full coil. The black dashed lines outline the myosepta between two somites to show the overall shape, and the black arrowheads indicate other visible myosepta boundaries.
Figure 4
Figure 4
The average measurements of morphological features of 6 dpf embryos over-expressing Δ95–98 kcnj2-12, WT kcnj2-12, and combined Δ95–98 and WT kcnj2-12 proteins. (a) The average distance between the hyosymplectic cartilages. (b) The average width of the Meckel's cartilage. (c) The average pupil-to-pupil distance. The results were averaged over three experiments and the error bars represent the standard error for the means. The P values were calculated using one-way ANOVA. * P < 0.05, *** P < 0.001.
Figure 5
Figure 5
Representative images of bone and cartilage staining of 6 dpf embryos expressing Δ95–98 kcnj2-12, WT kcnj2-12, and combined Δ95–98 and WT kcnj2-12 proteins. Black arrowheads show nonuniform eye development; red arrowheads show a protruding jaw; and yellow arrowheads show deformities in the side of the jaw. Scale bars: 100 μm.
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