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Dis Model Mech
2013 May 01;63:652-60. doi: 10.1242/dmm.009480.
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Generation and validation of a zebrafish model of EAST (epilepsy, ataxia, sensorineural deafness and tubulopathy) syndrome.
Mahmood F
,
Mozere M
,
Zdebik AA
,
Stanescu HC
,
Tobin J
,
Beales PL
,
Kleta R
,
Bockenhauer D
,
Russell C
.
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Recessive mutations in KCNJ10, which encodes an inwardly rectifying potassium channel, were recently identified as the cause of EAST syndrome, a severe and disabling multi-organ disorder consisting of epilepsy, ataxia, sensorineural deafness and tubulopathy that becomes clinically apparent with seizures in infancy. A Kcnj10 knockout mouse shows postnatal mortality and is therefore not suitable for drug discovery. Because zebrafish are ideal for in vivo screening for potential therapeutics, we tested whether kcnj10 knockdown in zebrafish would fill this need. We cloned zebrafish kcnj10 and demonstrated that its function is equivalent to that of human KCNJ10. We next injected splice- and translation-blocking kcnj10 antisense morpholino oligonucleotides and reproduced the cardinal symptoms of EAST syndrome - ataxia, epilepsy and renal tubular defects. Several of these phenotypes could be assayed in an automated manner. We could rescue the morphant phenotype with complementary RNA (cRNA) encoding human wild-type KCNJ10, but not with cRNA encoding a KCNJ10 mutation identified in individuals with EAST syndrome. Our results suggest that zebrafish will be a valuable tool to screen for compounds that are potentially therapeutic for EAST syndrome or its individual symptoms. Knockdown of kcnj10 represents the first zebrafish model of a salt-losing tubulopathy, which has relevance for blood pressure control.
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23471908
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Fig. 1. Identification and function of ZF kcnj10. (A) The 5â² end of ZF kcnj10a cDNA. The start codon and the first upstream stop codon are highlighted. (B) Alignment of the Kcnj10a and Kcnj10b protein sequences from ZF (DrJ10A and DrJ10B) and human (HsJ10) shows the high similarity between the orthologues. Black shows sequences that are identical in all three proteins; grey shows sequences that are identical in two of the proteins. (CâE) Heterologous expression of ZF Kcnj10a in Xenopus laevis oocytes results in K+-selective currents. Currents were obtained by two-electrode voltage clamp with voltage steps from â100 to +60 mV with 98 mM KCl (C) or 2 mM KCl (D) in the bath solution and were superimposed. C and D depict typical experiments, whereas the averaged current-voltage (IâV) traces from all data are shown in E, with data from oocytes injected with water or human KCNJ10 for comparison. These current-voltage traces reflect the average currents obtained 50 ms after the cells were clamped to the voltage steps indicated above. Currents were nonlinear, i.e. inwardly rectified, in keeping with the properties of currents mediated by human KCNJ10. This characteristic property is attributed to pore block by intracellular cations, which only affects outward currents. Ba, barium.
Fig. 2. ZF kcnj10a is expressed in brain, otic vesicles and pronephros. At 24 hpf, kcnj10a expression cannot be detected by in situ hybridisation (not shown) but, by 48 hpf (A), kcnj10a is expressed strongly in the mid-hindbrain boundary (MHB) and caudal hindbrain (cHb), and weakly in the midbrain and rostral hindbrain. (A–F) Until 120 hpf, kcnj10a expression becomes stronger, especially in the midbrain and rostral hindbrain (see Mb and rHb labels in B), and in the cerebellum (cp in E). More posteriorly, expression can be seen in the spinal cord (sc in F), but there is no evidence of expression in the lateral line. (I–O) Transverse sections at 120 hpf demonstrate kcnj10a expression in the inner nuclear layer of the retina (r in I) and reveal the majority of midbrain (J), hindbrain (K,L,M) and spinal cord (N) expression to be adjacent to the ventricles (for example, v in J). kcnj10a is also expressed in the cerebellum (cp in E,K), otic vesicle (ov in L,M) and weakly in the pronephros (p in G,H,O). (A–H) Whole-mount embryos with H being a higher magnification image of the pronephros (p) shown in G. (A–D,F) Lateral views with anterior to the left and dorsal up. (E,G,H) Dorsal views with anterior to the left. (I–L) Progressively more posterior transverse sections. (M–O) Higher magnification images of the otic vesicle, spinal cord and pronephros. ot, optic tectum. Scale bars: 100 μm.
Fig. 3. kcnj10a morphant ZF display movement defects. (A) Morphants showed an increased frequency of spontaneous contractions at 30 hpf, and this increase could be rescued by co-injection of normal human KCNJ10 cRNA, but not R65P mutant cRNA (n=10â15 per experimental condition). (B) At 120 hpf, we observed abnormal movements with a significantly higher frequency of jaw, eye and fin movements in kcnj10a morphants (black bars, n=7) than in controls (white bars, n=5). (C,D) Shown are 3-minute tracks from four WT (C) and four kcnj10a morphant (D) ZF at 120 hpf after being startled by touch. kcnj10a morphants (D) exhibit circling locomotion with frequent âloopsâ around their vertical axis. *P<0.05.
Fig. 4. kcnj10a morphants have kidney defects. (A,B) The upper images shows three 120-hpf WT larvae (A) and three kcnj10a morphants (B), some of which have pericardial edema. The lower image is an enlargement of the swim bladder and pronephric duct area. In morphants, the pronephric duct is visible because it is dilated (dotted arrow) and, although partially obscured by pigment, the swim bladder is not visible (arrowhead). (C,D) The upper and lower images show a 72-hpf WT (C) and a kcnj10a morphant (D) immediately after fluorescent dextran injection (upper image) and 24 hours later (lower image). Although baseline fluorescence immediately after injection was comparable in WT and morphant, there was significantly higher remaining fluorescence after 24 hours in morphant (D) compared with WT (C) ZF. (E) Graph of measured fluorescence in WT (triangles; n=9) and morphant ZF (squares; n=12). y-axis shows fluorescence intensity normalised for the baseline measurement in WT. x-axis shows time (hours).
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