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Figure 3. Cilia motility is impaired in Xenopus embryos depleted of components of the RNF20 complex. (A) Cilia beat frequency (CBF) in Ctrl and rnf20 MO LRO explants. Cilia were visualized by expressing Arl13b-GFP. Time-lapse montage of a single cilium. The space between the red arrows represents the number of frames of a ciliary single cycle. (B) Quantification of CBF in the LRO in Ctrl and rnf20 MO-injected embryos. Unpaired t test, ****P < 0.00005. (C, Top) Lateral view of an embryo with the epidermal multicilia labeled with antiacetylated tubulin and F-actin stained with phalloidin. (C, Bottom) TEM of Xenopus Ctrl and rnf20 morphant embryo epidermal cilia. IDAs are indicated by blue arrowheads. (D, Top) Analysis of epidermal cilia motility by tracking the fluorescent bead trajectory on the epidermal surface. Maximum projection of time-lapse images obtained during 0.3 s demonstrates the distance traveled by a bead propelled by motile cilia on the surface of the embryo. (D, Bottom) Quantification of bead velocity in Ctrl and rnf20 MO embryos at stages 22 and 26. n, number of embryos examined; 10–15 different beads per embryo were followed. Unpaired t test, ***P < 0.0005. (E) Quantified bead velocity after suboptimal dose MO (1/2), rnf20 (r20), rnf40 (r40), full-dose ube2b, or coinjected suboptimal doses of MO. n, number of embryos from three independent experiments. χ2/Fisher’s exact test, ****P < 0.00005, **P < 0.005. ns, not significant. (F) Percentage of embryos with abnormal pitx2c expression after suboptimal dose MO (1/2), rnf20, rnf40, full-dose ube2b, or coinjected suboptimal doses of MO. n, number of embryos from three independent experiments. χ2/Fisher’s exact test, ****P < 0.00005, *P < 0.05. Error bars in D and E are SD.

Image published in: Robson A et al. (2019)

Copyright © 2019. Image reproduced with permission of the Publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.

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