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. 2023 Jan 26;34(4):590–606. doi: 10.1681/ASN.0000000000000082

Figure 2.

Figure 2

Loss of Grk4 impacts on cilia formation and function in the zebrafish pronephros. (A) Grk4 morphant pronephric tubules are dilated compared with tubules of control-injected zebrafish, but coinjection of grk4 RNA reduces dilatation efficiently. Confocal z-stacks of 48 hpf zebrafish pronephric tubules. Green, anti-PKCζ staining apical cell borders of the tubule; red, anti-acTub. to visualize cilia. Scale bars: 10 µm. Graph shows n=23 (CTRL MO), 21 (Grk4 MO), and 34 (Grk4 MO+grk4) embryos. Kruskal-Wallis test with Dunn multiple comparison test. ****P< 0.0001. (B) Individual cilia from distal ends of pronephric tubules of injected zebrafish are longer after depletion of Grk4. Scale bar: 5 µm. Graph summarizes n=151 (CTRL MO) and 179 (Grk4 MO) cilia. Two-tailed Mann-Whitney test, ****P< 0.0001. (C) Grk4-depleted embryos clear fluids more slowly than control embryos. Representative images of embryos injected with FITC-dextrane into the pericardiac cavity shortly after injection (T1) and 24 hours later. Graphs depict disappearance of fluorescence in pericardiac cavity 0.5 (T1), 3 (T2), 5 (T3), and 24 hours postinjection (T4) in individual fish. P =0.0387, Mann-Whitney test. (D) Western blot of HEK cells transiently transfected with Grk4 wt or Grk4 mutated to match the zebrafish Cas9-edited allele, demonstrating lower expression of mutant Grk4, compared with wt. GAPDH as loading control. (E) pronephric tubules stained as in (A) of 48 hpf wt and grk4-mutant embryos showing a similar dilatation as in knockdown embryos. Scale bars: 10 µm. Left graph shows proximal pronephros diameter in wt and Grk4 mutant embryos at 48 hpf. n=14 (grk4+/+) and 15 (grk4mut/mut) embryos. Two-tailed Welch test. **P=0.0030. Graph on right displays cilia measurements in the distal pronephros of Grk4 mutants. n=124 (grk4+/+) and 123 (grk4mut/mut) cilia from 4 to 5 embryos, respectively. Two-tailed Welch test. ****P< 0.0001. All graphs: Red line indicates median. Circles represent individual experiments. acTub., acetylated tubulin; PKC, protein kinase C.