miR-34b regulates kidney morphogenesis by controlling multiciliogenesis and fluid flow. (A) Heat map showing the expression of genes in the GO term Cilium Assembly. The color scale represents the expression level of a gene above (red) or below (green) the mean expression level across all samples. (B) QRT-PCR analysis of the expression of cilia genes in MCCs that were isolated from 3-dpf Tg(cmyb:EGFP) embryos by flow cytometry. The expression levels were normalized to that of β-actin 1. (C) The ciliary (anti-acetylated tubulin staining, red, arrows) morphology of the MCCs (anti-GFP staining, green) at 36 hpf in normal embryos and miR-34b morphants. Arrows indicate normal cilia bundles. (D) The percentage of cmyb:EGFP+ cells with cilia bundles was calculated for each embryo and analyzed statistically at 36 hpf. (E) The ciliary morphology of the olfactory placode (outlined) at 3 dpf in normal embryos and miR-34b morphants. The number of embryos with normal cilia (top picture) or shorter cilia (bottom picture) among all analyzed embryos is indicated (bottom left). (F) Representative example of the dye excretion assay (3.5-dpf normal embryo), in which the time from injection until the first visible urine excretion at the cloaca (arrow) is measured. (G) Statistical analysis of the time from injection to dye excretion. Each circle/triangle represents an embryo. Mean ± s.d. ***P<0.0001 (unpaired t-test). Scale bar: 10 μm.
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