Knockdown of Kif15 with splicing-blocking method inhibits the normal formation of vestibular. (A,B) The protein level of Kif15 is severely decreased in Kif15-morpholino (MO)-injected embryos both in the band intensity (A) and in the semiquantitative analysis (B), in comparison with control embryos. Data are recorded as mean ± SEM. **p < 0.01. (C–H) Kif15-MO suppresses the normal development of semicircular canals. The abnormal manifestations are observed at 48 hpf (C,F), 72 hpf (D,G), and 96 hpf (E,H), respectively. ap, anterior protrusion; ab, anterior bulge; vb, ventral bulge; vp, ventral protrusion; pb, posterior bulge; pp, posterior protrusion; u, utricle; and s, saccule. Red arrowheads show the abnormal fusion of anterior and posterior protrusions. (I–K) Co-injection using Kif15-MO and Kif15 mRNA successfully rescues the phenotypes of semicircular canals and otoliths at 48 hpf (I), 72 hpf (J), and 96 hpf (K), respectively. Scale bar is 20 μm.

Knockdown of Kif15 disturbs the normal development of otolith organs. (A–E) Kif15 inhibition causes abnormal phenotypes of otoliths, including multiplication (B) and reduction (E) in number, and two abnormal otoliths (C,D) at 48 hpf. (F) Co-injection Kif15 mRNA with Kif15-MO successfully rescues the phenotypes of otolith organs at 48 hpf. (G) Different types of abnormal otoliths and the corresponding proportion analysis in Kif15-MO-injected embryos (n = 234), untreated controls (n = 136), and Kif15-MO + Kif15 mRNA group (n = 111). Scale bar is 20 μm.

Kif15 inhibition results in decreased hair cells (HCs) in the inner ear. (A–C) The gross morphology of otic vesicle in different groups at 48 hpf using the white light field microscope. u, utricle; s, saccule. Scale bar is 20 μm. (D–F) Representative images of utricular HCs in different groups at 48 hpf. (G–I) Representative images of saccular HCs in different groups at 48 hpf. White star labels the HC. (J) Quantification of the number of HCs in utricle and saccule in control-MO (Con-MO) group (n = 22), Kif15-deficient morphants (n = 22), and Kif15-MO + Kif15 mRNA group. Data are recorded as mean ± SEM. **p < 0.01, ***p < 0.001 vs. Con-MO group; ^##p < 0.01 vs. Kif15-MO group; NS, no significance.

Kif15 knocking down leads to abnormal posterior lateral line (PLL) development, which is represented by a decrease in the number of neuromasts (NMs). (A–D) Kif15 inhibition induces the reduction of NMs in zebrafish PLL, while co-injection of Kif15 mRNA with Kif15-MO successfully rescues the decreased number of NMs compared to the Con-MO-treated controls. The PLL NMs are indicated by white arrowheads. Scale bar is 200 μm. (E) Statistical analysis in quantification of PLL NMs in Con-MO-injected controls, Kif15-MO-injected embryos, and Kif15-MO + Kif15 mRNA group (n = 50 in the three groups). Data are recorded as mean ± SEM. *p < 0.05, ****p < 0.0001 vs. Con-MO group; ^####p < 0.0001 vs. Kif15-MO group. (F) Simple and intuitive schematic diagram of changes in PLL development after Kif15 knockdown by MO.

The activity and behavior of zebrafish are abnormal after knocking down Kif15. (A) The locomotion assay of zebrafish in Con-MO (n = 32), Kif15-MO (n = 31), and Kif15-MO + Kif15 mRNA (n = 30) injected larvae in traced locomotion curve. (B) The moved distance from the original point is analyzed and compared among different groups. The unit of the Y-axis is mm. Data are recorded as mean ± SEM. **p < 0.01, ***p < 0.001 vs. Con-MO group; ^#p < 0.05 vs. Kif15-MO group.