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Figure 2. (a) RT–PCR demonstrates disrupted jbts17 splicing after MO injection (Jbts17-KD). odc1 is the loading control. (b) In situ hybridization of the Sonic Hedgehog (SHH) direct target nkx2.2 in control embryos and ones with Jbts17 knockdown (stage 22). Scale bars, 2 mm. Fractions represent the fraction of embryos displaying the phenotype. (c) Expression of pitx2, which labels the left lateral plate, at stage 26. Arrows indicate signal in left lateral plate mesoderm (LPM); the graph represents pitx2 expression patterns in embryos. Scale bars, 1 mm. (d) Immunostaining for acetylated α-tubulin, which labels cilia, shows ciliogenesis defects after Jbts17 knockdown in the ventral neural tube (stage 22). Scale bars, 10 μm. (e) Immunostaining for acetylated α-tubulin shows that Jbts17 knockdown reduces cilia length; cilia numbers are unchanged. Scale bars, 10 μm; membrane-RFP labels membranes. The graphs in d and e each show pooled data from two independent experiments for cilia length (shown as means ± s.e.m.; ***P < 0.001). (f) MCCs from control embryos, embryos with Jbts17 knockdown showing disrupted cilia, and Jbts17-knockdown embryos rescued with untargeted jbts17 mRNA labeled by GFP-Cfap20 (green); membrane-RFP labels membranes. Scale bars, 10 μm. (g) GFP-tagged Jbts17 localizes near basal bodies (visualized with coexpressed Cetn4-RFP) in an MCC. Scale bar, 10 μm. (h) Super-resolution image of GFP-Jbts17 and mCherry-Cep164 at a single basal body; both form rings of ~260 nm in diameter around the basal body, visualized by Cetn4-BFP. Diameters are shown as means ± s.d. in each panel. The graph shows fluorescence intensities (in arbitrary units) for GFP-Jbts17, mCherry-Cep164 and Cetn4-BFP. Scale bar, 100 nm. (i) GFP-tagged CPLANE proteins (green) and basal bodies visualized by Cetn4-RFP (magenta) in control and Jbts17-knockdown MCCs. Scale bars, 1 μm. In box plots of CPLANE fluorescence intensities at basal bodies, boxes extend from the 25th to the 75th percentile, with a line at the median; whiskers indicate maximum and minimum values. **P = 0.0041, ***P < 0.001; NS, not significant. Con, control. (j) Table summarizing the localization of CPLANE proteins at basal bodies for each knockdown (Supplementary Fig. 2c–f). |
