Cilium structure is normal and maintained with age in Root mutant neurons, and centrioles but not cilia are required for rootlet assembly. (A) Various ciliated neurons at indicated age expressing mCD8-GFP to label ciliary membranes and Cby-Tomato to mark the transition zone (Tz). Brackets indicate the cilia, and arrows the Tz. Cilium morphology appears normal in Root⁶⁶ through aging. hot/cold cells: temperature-sensing neurons in the arista, olfactory: olfactory neurons in the antennal third segment. For the hot/cold neuron images, transmitted light images are overlaid to show the morphology of the arista. (B) Olfactory neurons stained for 21A6 to label the cilium base and Cby-Tom to label the Tz. Cby and 21A6 localizations appear normal in Root⁶⁶. (C) Ch neurons in the JO stained for actin to label the scolopale rods and 21A6 to label cilia. 21A6 localizes both to the cilium base and a distal region in the cilium (arrows), and this localization appears normal in Root⁶⁶. (D) Cross-section of JO cilia by transmission electron microscopy shows that Root⁶⁶ axoneme ultrastructure appears normal. (E) Sas-4 mutant lacking centrioles fails to organize rootlets in olfactory neurons. Compared with the control, where rootlets project from cilium base marked by 21A6 (arrows), rootlet structures are absent in most Sas-4 olfactory neurons (solid arrowheads), although sometimes abnormal tiny fibers are associated with 21A6 (open arrowheads). Endogenous Root is stained in green. (F) In control olfactory neurons, all rootlets are associated with the cilium base marker 21A6 (empty arrows), and the basal bodies (dBB and pBB) marked by Ana1-GFP are in tandem (solid arrows). In Plp mutant that lacks cilia and has the basal bodies displaced from the dendrite tip, most of the rootlets (empty arrowheads) are associated with a single basal body (solid arrowheads), which is not attached with 21A6. B, C, E, and F show images of the antenna squash. Bars: (A–C and E) 10 µm; (B, inset) 1 µm; (D) 200 nm; (F, main) 10 µm; (F, zoom) 5 µm; (F, zoom inset) 1 µm. See also Fig. S3.