zebrafish ik mutants display ciliopathy-like phenotypes (A) Whole-mount in situ hybridization (WISH) analysis of zebrafish ik mRNA at different developmental stages (1-cell, 4-cell, 256-cell, Germ-ring, Bud, 14 somites, 1 dpf, 2 dpf, 3 dpf, and 4 dpf). Scale bar, 250 μm (B) Brightfield microscopic images of the body curvature, otolith, and pronephric cysts pronephros in WT embryos and ik mutants (ik^−/−) at 2 dpf. Pronephric cyst of ik mutants is marked with red arrowhead. Stacked bar graph displays the percentage of embryos with a ventrally curved body, abnormal otolith phenotype, and cyst formation (C) WISH analysis of cmlc2 for the location of whole heart in WT embryos and ik mutants at 2 dpf (D) Schematic representation of the coding region of zebrafish ik. The target region (exon 2) of the morpholino employed in this study was represented using a blue line (E) RT-PCR analysis of ik mRNA expression in control morphants (MO), ik MO, and ik MO/ik mRNA co-injected embryos at 2 dpf. gapdh served as a normalization control and band intensity ratio of ik/gapdh mRNA expression was marked (F) Brightfield microscopic images of control MO, ik MO, and ik MO/ik mRNA co-injected embryos at 2 dpf. Whole body, otolith, and kidney cyst were captured. Pronephric cysts of ik MO are marked with red arrowhead

Loss of ik results in abnormal ciliary morphology (A) Whole-mount immunostaining of cilia in the inner ear (white boxes) using anti-acetylated-α-tubulin (green) of WT embryos (n = 3) and ik mutants (n = 5) at 2 dpf. Scale bar, 20 μm. Quantified graphs of cilia lengths and numbers in the inner ear between WT embryos and ik mutants at 2 dpf. *p < 0.05, ***p < 0.001 (B) Confocal microscopic images of control MO, ik MO, and ik MO/ik mRNA co-injected Tg(brn3c:GFP) embryos at 3 dpf. Scale bar, 20 µm. Quantification of ciliary length and number in the inner ear of control MO (n = 12), ik MO (n = 17), and ik MO/ik mRNA co-injected embryos (n = 10). **p < 0.01, ***p < 0.001 (C) Whole-mount immunofluorescence (left) of overall cilia in the pronephros in WT and in ik mutants at 2 dpf using anti-acetylated-α-tubulin (green). Scale bar, 100 µm. Enlarged view (right) of pronephric cilia in proximal and distal tubules stained with anti-acetylated-α-tubulin (green) and nuclei stained with DAPI (blue) at 2 dpf WT embryos and ik mutants. Scale bar, 20 µm. Stacked bar graph displays the percentage of embryos of cilia phenotype in the anterior pronephric duct of WT embryos and ik mutants at 2 dpf. (D) TEM results showing the ultrastructure of cilia in the pronephric duct of ik mutants. Cross-section showing the “9 + 2” configuration (black circles). Scale bar, 100 nm (E) Whole-mount immunofluorescence (left) of overall cilia in the pronephros of control MO, ik MO, and ik MO/ik mRNA co-injected embryos at 3 dpf with anti-acetylated-α-tubulin (green). Scale bar, 100 µm. Enlarged view (right) of pronephric cilia in the anterior and posterior pronephric ducts stained using anti-acetylated-α-tubulin (green) and nuclei stained with DAPI (blue) at 2 dpf control MO, ik MO, and ik MO/ ik mRNA co-injected embryos. Scale bar, 20 µm. Stacked bar graph displays the percentage of embryos based on ciliary morphology in the anterior pronephric duct (percentage of abnormal ciliary morphology of embryos: control MO, 0%; ik MO, 58%; ik MO + ik mRNA, 0%)

ik mutants show impaired kidney development with abnormal cilia morphology (A) WISH analysis of pronephric duct markers slc4a4, slc13a1, and slc12a3 in WT embryos and ik mutants at 2 dpf. Scale bar, 250 μm (B) RT-PCR analysis of slc4a4, slc13a1, and slc12a3 expression in WT embryos and ik mutants at 2 dpf. gapdh served as a normalization control. Relative bands intensities of slc4a4, slc13a1, and slc12a3 normalized to gapdh intensity was graphically represented (in triplicates) using Image J software. **p < 0.01, ***p < 0.001 (C) Linear filter model showing correlation of overall gene expression based on RNA-seq data between WT and ik mutants at 3 dpf. The point of foxj1a plot is indicated. (D) RT-PCR analysis of foxj1a expression in WT embryos and ik mutants at 1.5 and 2.5 dpf. gapdh served as a normalization control. Relative band intensity of foxj1a normalized with respect to gapdh intensity is graphically represented (in triplicates) *p < 0.05, **p < 0.01 (E) WISH analysis of foxj1a in WT and ik mutants at 1.5, 2, and 2.5 dpf. Scale bar, 250 μm (F) WISH analysis of foxj1a in control MO, ik MO, and ik MO/ik mRNA co-injected embryos at 2.5 dpf. Scale bar, 250 μm

ofd1, a necessary gene for cilia assembly, is downregulated by the loss of ik (A) Heat map of seven differentially expressed cilium assembly genes at least 1.5 > log2 fold changes in ik mutants relative to those of WT embryos. Upregulated DEGs relative to the mean are indicated by red color. Downregulated DEGs are shown by blue color (scale bar, log 2 of mRNA ratio) (B) RT-PCR analysis of ofd1 mRNA expression in WT embryos and ik mutants. gapdh served as a normalization control. Relative band intensity of ofd1 normalized with respect to gapdh intensity is graphically represented (in triplicate) using Image J software. **p < 0.01 (C) WISH for ofd1 in WT embryos and ik mutants at 1 dpf. Scale bar, 100 μm. Enlarged view of the pronephros in WT and ik mutants at 1 dpf. Scale bar, 150 μm (D) RT-PCR analysis of ofd1 mRNA expression in control MO, ik MO, and ik MO/ik mRNA co-injected embryos at 1.5 dpf. gapdh served as a normalization control. Relative band intensity of ofd1 normalized with respect to gapdh intensity is graphically represented (in three individual experiments) using Image J software. **p < 0.01, ***p < 0.001 (E) WISH for ofd1 in control MO, ik MO, and ik MO/ik mRNA co-injected embryos at 1 dpf. Enlarged view of the pronephros in control MO, ik MO, and ik MO/ik mRNA co-injected embryos at 1 dpf. Scale bar, 100 μm

The ciliopathy phenotypes of ik mutants are rescued by ofd1 co-injection (A) The lateral morphological view of control MO, ik MO, and ik MO/ofd1 mRNA co-injected embryos (body curvature and otolith imaged at 2 dpf and pronephric cyst imaged at 3 dpf). Pronephric cyst in ik MO is indicated by red arrowhead. Stacked bar graph displays the phenotypic percentage of embryos. B WISH for ofd1 in control MO, ik MO, and ik MO/ofd1 mRNA co-injected embryos at 1 dpf. Scale bar, 100 μm. Enlarged view of the pronephros in control MO, ik MO, and ik MO/ik mRNA co-injected embryos at 1 dpf. Scale bar, 150 μm. C WISH for foxj1a in control MO, ik MO, and ik MO/ofd1 mRNA co-injected embryos at 2.5 dpf. Scale bar, 100 μm (D) RT-PCR analysis of foxj1a mRNA expression in control MO, ik MO, and ik MO/ofd1 mRNA co-injected embryos at 1.5 dpf. gapdh served as a normalization control. Relative band intensity of foxj1a normalized with respect to gapdh intensity is graphically represented using Image J software (in three individual experiments). *p < 0.05, ***p < 0.001

ofd1 co-injection can rescue ciliary dysmorphology of ik morphants (A) Confocal microscopic images of control MO, ik MO, and ik MO/ofd1 mRNA co-injected Tg(brn3c:GFP) embryos at 2 dpf. Scale bar, 20 µm. Bar graphs show quantification of ciliary length and number in the inner ears of control MO, ik MO, and ik MO/ofd1 mRNA co-injected embryos at 2 dpf. **p < 0.01, ***p < 0.001 (B) Whole-mount immunostaining of pronephric cilia using anti-acetylated-α-tubulin (red) at 2 dpf control MO, ik MO, and ik MO/ ofd1 mRNA co-injected embryos. Scale bar, 20 µm. Stacked bar graph displays the percentage of ciliary morphology of embryos in the anterior pronephric duct (percentage of abnormal ciliary morphology of embryos: control MO, 5.12% (37/39); ik MO, 86.6% (4/30); ik MO + ofd1 mRNA, 16.6% (15/18)