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Fig. 3

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ZDB-IMAGE-160510-18
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Image
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Fig. 3

Forced nuclear localization does not re-establish the transcriptional activity of GLIS2/NPHP7C175R. (a) A consensus nuclear localization sequence (NLS) fused to the amino terminus re-establishes the nuclear localization of GLIS2/NPHP7C175R. (b) Although wild-type GLIS2/NPHP7 activates the mouse insulin-2 promoter, both GLIS2/NPHP7C175R and GLIS2/NPHP7C175R fused with a NLS fail to activate this promoter luciferase construct. (c) Wild-type-like pronephric morphology of 48 -h-old zebrafish embryo injected with control morpholino oligonucleotide (MO). In these experiments, the wt1b:GFP transgenic zebrafish line that labels the glomeruli and the proximal tubules was used. (d) In contrast, nphp7.2 MO injected embryos develop pronephric cysts, evident in the dilation of the glomeruli and the proximal tubules (outlined in white). (e) Knockdown of zebrafish nphp7.2 with splice-blocking MOs (0.1 mm)9 results in pronephric cysts. Co-expression of zebrafish nphp7.2 mRNA (300 ng/µl; WT RNA) rescues the phenotype and reduces cyst formation. In contrast, the corresponding nphp7.2 mutation (C175R RNA; 300 ng/µl) fails to rescue the phenotype, and instead slightly increases cyst formation. Neither wild-type nor mutant nphp7.2 mRNA displays a significant phenotype, if co-expressed with a control MO. The black bars represent the average percentages of cyst formation from five independent experiments (N). The numbers within the bars represent the total number of embryos analyzed (n). To calculate the depicted P-values, the Cochran-Mantel-Haenszel test was used. For complete statistical analysis, see Supplementary Materials. Scale bar, 100 µm in c and d.

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