Figure 2. Jbts17 localizes to the base of cilia and is required for ciliogenesis and cilia-mediated patterning.

(a) RT-PCR demonstrates disrupted Jbts17 splicing after MO injection. ODC1 = loading control. (b) In situ hybridization of SHH direct target, nkx2.2 in Jbts17-knockdown ( st. 22). (c) Pitx2 expression at st. 26. Arrows indicate signal in left lateral plate mesoderm (LPM); graph indicates pitx2 expressing embryos. (d) Acetylated α-tubulin immunostaining in the ventral neural tube (st. 22)(Scale bars, 10 μm). (e) Nodal cilia length is reduced; cilia numbers are unchanged. (Scale bars, 10 μm). Graphs in D and E e a c h show pooled data from two independent experiments c i l i a length (mean +/- SEM; ***p <0.001). (f) Multi-ciliated cells in control, Jbts17-knockdown (Jbts17-KD), and rescue with untargeted Jbts17 mRNA. Cilia and cell membrane visualized by GFP-CFAP20 (green) and membrane-RFP (magenta). Scale bars, 10 μm. (g) GFP-tagged Jbts17 localizes near basal bodies (visualized by co-expressed centrin4-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 ~ 260nm around the basal body, visualized by centrin4-BFP. Diameters shown as mean ± SD in each panel. The lower graph shows fluorescence intensities for GFP-Jbts17, mCherry-Cep164, and centrin4-BFP (Scale bar, 100 nm). (i) GFP-tagged CPLANE proteins (green) and basal bodies visualized by centrin4-RFP (magenta) in control and Jbts17 knockdown multi-ciliated cells; box plots of CPLANE intensities at basal bodies; boxes extend from 25^th-75th percentiles, with a line at the median; whiskers indicate max and min. (j) Table summarizes the localization of CPLANE proteins at basal bodies for each knockdown (see Supplementary Fig. 2c-f).