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The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear


In teleosts, proper balance and hearing depend on mechanical sensors in the inner ear. These sensors include actin-based microvilli and microtubule-based cilia that extend from the surface of sensory hair cells and attach to biomineralized ‘ear stones’ (or otoliths)1. Otolith number, size and placement are under strict developmental control, but the mechanisms that ensure otolith assembly atop specific cells of the sensory epithelium are unclear. Here we demonstrate that cilia motility is required for normal otolith assembly and localization. Using in vivo video microscopy, we show that motile tether cilia at opposite poles of the otic vesicle create fluid vortices that attract otolith precursor particles, thereby biasing an otherwise random distribution to direct localized otolith seeding on tether cilia. Independent knockdown of subunits for the dynein regulatory complex and outer-arm dynein disrupt cilia motility, leading to defective otolith biogenesis. These results demonstrate a requirement for the dynein regulatory complex in vertebrates and show that cilia-driven flow is a key epigenetic factor in controlling otolith biomineralization.

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Figure 1: gas8 is expressed in ciliated tissues.
Figure 2: gas8 morphants exhibit developmental defects.
Figure 3: Gas8 is required for tether cilia motility.
Figure 4: Tether cilia motility drives otolith biogenesis.


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We are grateful to R. Crosbie for discussions and encouragement throughout the course of the project. We thank I. Drummond and C. Nguyen for discussions and comments on the work. We are grateful to L. Trinh, O. Bricaud and A. Collazo for sharing reagents and for providing probes, as well as to all the members of the Fraser laboratory for discussions, in particular M. Liebling and W. Supatto for sharing Matlab scripts and comments. We are grateful to Z. P. Kabututu for performing the lrrc50 reverse transcriptase PCR experiments. J.V. was supported by a fellowship from the Human Frontier Science Program, D.W. was supported by the NIH Medical Scientist Training Program at UCLA/Caltech. J.R.C. was supported by NIH RSDA training grant no. M07185 and a Warsaw Fellowship from the MIMG Department at UCLA. A.D.L is supported by an NSF fellowship. This work was supported by NIH grant R01 HL081799 (J.C.), NIH grant R01AI52348 and Beckman Young Investigator Award (K.L.H.).

Author Contributions J.R.C., J.V., D.W. and K.L.H. designed the experiments and interpreted the results. J.R.C, J.V. and D.W. conducted the experiments. J.V. and D.W. developed the equipment and systems for and performed in vivo video imaging for the quantitative flow study and analyzed the data with J.R.C., S.F. and K.L.H. A.D.L. assisted with in situ hybridization. A.D.L. and J.C. provided guidance on gas8 morpholino injections. The manuscript was written by J.R.C., J.V., D.W. and K.L.H. All authors discussed the results and commented on the manuscript.

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Correspondence to Kent L. Hill.

Supplementary information

Supplementary Figures and legends

This file contains Supplementary Figures S1-S6 with legends, and legends for Supplementary Movies 1-11b. (PDF 2347 kb)

Supplementary movie 1

Supplemental movie 1. Three dimensional display of a control inner ear at stage 27 hpf after immunofluorescence labelling of cilia with acetylated tubulin antibody. (MOV 1270 kb)

Supplementary movie 2

Supplemental movie 2. Three dimensional display of a gas11 morphant inner ear at stage 27 hpf after acetylated tubulin immunofluorescence labelling. (MOV 1304 kb)

Supplementary movie 3

Supplemental movie 3. Video shows tether cilia motility in control embryo. (MOV 1303 kb)

Supplementary movie 4

Supplemental movie 4. Video shows tether cilia motility in control embryo. (MOV 1496 kb)

Supplementary movie 5

Supplemental movie 5. Video shows tether cilia motility in control embryo. (MOV 2952 kb)

Supplementary movie 6

Supplemental movie 6. Video shows short cilia in control embryo are not motile. (MOV 5902 kb)

Supplementary movie 7

Supplemental movie 7. Video shows tether cilia in a gas11 morphant embryo are not motile. (MOV 831 kb)

Supplementary movie 8

Supplemental movie 8. Video shows tether cilia in a gas11 morphant embryo are not motile. (MOV 8050 kb)

Supplementary movie 9a

Supplemental movie 9a. High speed video microscopy of tether cilia in a control embryo at 24 hpf showing vortices in the vicinity of the tether cilia and particle propelling along the growing otolith. (MOV 1690 kb)

Supplementary movie 9b

Supplemental movie 9b. Same as 9a with particle tracking. (MOV 10688 kb)

Supplementary movie 10a

Supplemental movie 10a. High speed video microscopy of the inner ear in a control embryo at 23 hpf showing high displacement of otolith precursors in the vicinity of tether cilia and low displacement away from tether cilia. (MOV 3109 kb)

Supplementary movie 10b

Supplemental movie 10b. Same as supplemental movie 10a with particle tracking. (MOV 1591 kb)

Supplementary movie 11a

Supplemental movie 11a. High speed video microscopy of tether cilia in a gas11 morphant at 25 hpf showing very low particle displacement in the vicinity of tether cilia. (MOV 1357 kb)

Supplementary movie 11b

Supplemental movie 11b. Same as 11a with particle tracking. (MOV 14789 kb)

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Colantonio, J., Vermot, J., Wu, D. et al. The dynein regulatory complex is required for ciliary motility and otolith biogenesis in the inner ear. Nature 457, 205–209 (2009).

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