Cilia length and Hedgehog signaling are not impaired in the rare cilia formed in Hyls1 D226G MEFs. (A) Quantification of centrosome (γ-TUBULIN) and distal appendages (CEP164) from immunofluorescence images of Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG MEFs. MEFs generated across N ≥ 3 mice per genotype were analyzed. (B) Cilia length analysis of Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG MEFs. MEFs from N ≥ 3 mice per genotype were analyzed. (C) Quantification of Smoothened recruitment to cilia after SAG treatment from immunofluorescence images of Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG MEFs. MEFs from N ≥ 3 mice per genotype were analyzed. (D) Representative immunofluorescence images of the centrosome (γ-TUBULIN), Smoothened, and cilia (Ac-TUBULIN) in Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG MEFs. (E) Representative immunofluorescence images of the centrosome (γ-TUBULIN), FOXJ1, and cilia (Ac-TUBULIN) (left), and of the centrosome (γ-TUBULIN), centriole (CENTRIN2), and distal appendages (CEP164) (right) in Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG ependymal MCC. (F) Quantification of differentiated MCC abundance from immunofluorescence images of Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG ependymal cells in E. Cells generated from N ≥ 2 mice per genotype were analyzed. (G) U-ExM analysis of different stages of MCC differentiation in ependymal cells in vitro stained for centrioles and cilia (β-TUBULIN), CENTRIN2, and distal appendages (CEP164) in Hyls1^+/+, Hyls1^+/DG, and Hyls1^DG/DG ependymal cells. Data are represented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Tukey’s multiple comparisons test (A, B, and F). Only significant results are indicated. Smoothened analyses (C) were assessed using two-way ANOVA with post-hoc analysis, and results are summarized in supplementary material. (****) P < 0.0001. Scale bar is 5 μm in D and E and 500 nm in G. Inset diameter is 5.4 µm in D.