JNK activity is necessary for ciliogenesis. (A) Velocity of fluorescent beads in micrometers per second of control Xenopus embryos and embryos injected with JNK MO. MO (40 ng per cell) was injected into the ventral blastomeres at the four-cell stage. Scale bar, 500 μm. Quantification of the bead velocity shows impaired fluid flow in JNK morphants. Error bars indicate ± SEM from two experiments, n = 5 embryos (stage 29–30) per group. ***, P < 0.001, unpaired two-tailed t test. (B) Control embryos or JNK MO-injected embryos were fixed at stage 31/32 and stained for ac-tubulin and centrin. Coinjection of JNK MO with mScarlet-JNK1 rescued the ciliary defects. Scale bar, 10 μm. Quantifications of the acetylated tubulin intensity, percentage of ciliated, partially ciliated and non-ciliated cells (n = 305 control cells from three embryos, n = 398 JNK MO MCCs from four embryos, n = 431 from three embryos), and the percentage of cells displaying trapped basal bodies within the cytoplasm in controls (n = 203 MCCs from three embryos), JNK morphants (n = 580 from two embryos), and JNK MO/mScarlet JNK (n = 154 from three embryos) injected embryos. Apical cell surface area of epidermal MCCs is decreased in JNK MO-injected embryos. Error bars indicate ± SEM. **, P < 0.01, ***, P < 0.001, unpaired two-tailed t test. (C) Representative images of stage 30 control embryos or embryos injected with Flag-JBD under α-tubulin promoter. Embryos were either stained for acetylated tubulin or coinjected with RFP-Centrin to mark basal bodies. The graph shows a higher percentage of partially ciliated and non-ciliated cells (n = 288 MCCs from three embryos) as well as MCCs with defective BB migration in embryos injected with Flag-JBD compared with controls (n = 309 MCCs from three embryos). Error bars indicate ± SEM from three experiments. **, P < 0.01, ***, P < 0.001, unpaired two-tailed t test. Scale bar, 10 μm. (D) Quantification of the fluid flow with fluorescent beads shows decreased bead velocity in SP600125-treated embryos compared to DMSO. Error bars indicate ± SEM, n = 5 embryos (stage 29–30) per group. ****, P < 0.0001, unpaired two-tailed t test. Scale bar, 500 μm. (E) Confocal imaging of cilia in DMSO- and SP600125-treated stained with anti-acetylated tubulin and of basal bodies stained with anti-Centrin. Scale bars, 20 µm. Graph showing a higher percentage of partially ciliated and non-ciliated MCCs (n = 272 control cells from 5 embryos, n = 259 SP600125-treated cells from 6 embryos) and cells displaying defective basal body migration in SP600125-treated embryos (n = 118 from 5 embryos) compared with control (n = 120 from 5 embryos). Error bars indicate ± SEM from three experiments. *, P < 0.05, **, P < 0.01, unpaired two-tailed t test. (F) Western blot showing phospho-JUN levels in skin lysates from uninjected, multicilin overexpressing, and multicilin/JNK MO coinjected embryos. Quantification shows the normalized ratio of pJUN/GAPDH intensity. Data represent mean ± SEM from two independent experiments. *, P < 0.05, **, P < 0.01, unpaired two-tailed t test. Source data are available for this figure: SourceData F3.
Figure 3.

JNK activity is necessary for ciliogenesis. (A) Velocity of fluorescent beads in micrometers per second of control Xenopus embryos and embryos injected with JNK MO. MO (40 ng per cell) was injected into the ventral blastomeres at the four-cell stage. Scale bar, 500 μm. Quantification of the bead velocity shows impaired fluid flow in JNK morphants. Error bars indicate ± SEM from two experiments, n = 5 embryos (stage 29–30) per group. ***, P < 0.001, unpaired two-tailed t test. (B) Control embryos or JNK MO-injected embryos were fixed at stage 31/32 and stained for ac-tubulin and centrin. Coinjection of JNK MO with mScarlet-JNK1 rescued the ciliary defects. Scale bar, 10 μm. Quantifications of the acetylated tubulin intensity, percentage of ciliated, partially ciliated and non-ciliated cells (n = 305 control cells from three embryos, n = 398 JNK MO MCCs from four embryos, n = 431 from three embryos), and the percentage of cells displaying trapped basal bodies within the cytoplasm in controls (n = 203 MCCs from three embryos), JNK morphants (n = 580 from two embryos), and JNK MO/mScarlet JNK (n = 154 from three embryos) injected embryos. Apical cell surface area of epidermal MCCs is decreased in JNK MO-injected embryos. Error bars indicate ± SEM. **, P < 0.01, ***, P < 0.001, unpaired two-tailed t test. (C) Representative images of stage 30 control embryos or embryos injected with Flag-JBD under α-tubulin promoter. Embryos were either stained for acetylated tubulin or coinjected with RFP-Centrin to mark basal bodies. The graph shows a higher percentage of partially ciliated and non-ciliated cells (n = 288 MCCs from three embryos) as well as MCCs with defective BB migration in embryos injected with Flag-JBD compared with controls (n = 309 MCCs from three embryos). Error bars indicate ± SEM from three experiments. **, P < 0.01, ***, P < 0.001, unpaired two-tailed t test. Scale bar, 10 μm. (D) Quantification of the fluid flow with fluorescent beads shows decreased bead velocity in SP600125-treated embryos compared to DMSO. Error bars indicate ± SEM, n = 5 embryos (stage 29–30) per group. ****, P < 0.0001, unpaired two-tailed t test. Scale bar, 500 μm. (E) Confocal imaging of cilia in DMSO- and SP600125-treated stained with anti-acetylated tubulin and of basal bodies stained with anti-Centrin. Scale bars, 20 µm. Graph showing a higher percentage of partially ciliated and non-ciliated MCCs (n = 272 control cells from 5 embryos, n = 259 SP600125-treated cells from 6 embryos) and cells displaying defective basal body migration in SP600125-treated embryos (n = 118 from 5 embryos) compared with control (n = 120 from 5 embryos). Error bars indicate ± SEM from three experiments. *, P < 0.05, **, P < 0.01, unpaired two-tailed t test. (F) Western blot showing phospho-JUN levels in skin lysates from uninjected, multicilin overexpressing, and multicilin/JNK MO coinjected embryos. Quantification shows the normalized ratio of pJUN/GAPDH intensity. Data represent mean ± SEM from two independent experiments. *, P < 0.05, **, P < 0.01, unpaired two-tailed t test. Source data are available for this figure: SourceData F3.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal