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Fig. 3 Mci mutant MCC precursors are unable to generate multiple basal bodies. (A) A wild-type trachea section showing apically aligned multiple basal bodies in MCCs (arrows; stained using anti-pericentrin antibodies). (B) Section of Mci mutant trachea showing single basal body in monociliated cells (arrows). (C) TEM image showing multiple basal bodies (black arrows) and cilia (red arrows) in a wild-type MCC. Microvilli are also indicated (blue arrows). (D) TEM image showing lack of multiple basal bodies and cilia in a Mci mutant MCC. Microvilli are indicated (blue arrows). Five sections each from two independent wild-type and mutant tracheae were sampled, and the cells were examined from the basal to the apical end. We did not find any evidence of undocked basal bodies in the cytoplasm of Mci mutant cells. (E) Wild-type MCCs differentiated in ALI culture with multiple cilia (arrows). (F) Mci mutant airway cells differentiated in ALI culture with a single cilium (arrow). (G) Wild-type airway cells differentiated in ALI culture showing nuclear FOXJ1 expression. (H) Mci mutant airway cells differentiated in ALI culture showing nuclear FOXJ1 expression. (I) Wild-type MCCs differentiated in ALI culture with multiple basal bodies (stained with anti-γ-tubulin antibodies) and multiple cilia. (J) Display of only γ-tubulin staining from I. (K) Mci mutant cells differentiated in ALI culture with a single basal body (arrow) and a single cilium. Inset shows a single cilium and basal body (arrow). (L) Display of only γ-tubulin staining from K showing a single basal body (arrow). Inset shows a single basal body (arrow). In preparations shown in A,B,E,F,I,K, cilia were stained using anti-acetylated tubulin antibodies (green) and nuclei were stained with DAPI (blue). Scale bars: 5 μm. ALI cultures were carried out in three independent biological replicates.