WASP functions in the glial cells to regulate ciliogenesis in C. elegans neurons. (A) Schematic of the C. elegans amphid. Top: two glial cells, including the socket cell (AMso, pink) and the sheath cell (AMsh, blue). Each amphid has 12 neurons (only one is drawn in brown). The area in the rectangle is enlarged in the bottom. (B) Schematic of the sensory compartment3. Neuronal cilia are extended from dendritic endings and enter the sensory channel that is formed by the glial cells. TZ: transition zone. (C) The morphology of the amphid (top) and phasmid (bottom) cilia labeled with OSM-6::mCherry in WT and wsp-1a (gm324) mutant animals. Yellow arrowheads: defects of the ciliary distal segments in wsp-1a. White arrowheads indicate the boundaries of the ciliary distal and middle segments. Scale bar, 2 μm. (D) Quantifications of cilium phenotypes in WT and wsp-1a mutant animals. n = 50–80. (E) Kymographs (Middle (M) or Distal (D)) and corresponding lines (M' or D') of OSM-6::mCherry motility. Numbers represent mean ± SD (n = 80-112). Horizontal bar, 2 μm; vertical bar, 5 s. (F) Cross-section TEM analysis of the amphid channel cilia in WT and wsp-1a. Representative images show the cross sections of the transition zone, middle segment, distal segment and distal tip. Yellow asterisks: highly electron-dense materials. Yellow arrowheads: collapse of the channel. Scale bar, 500 nm. (G) Top (left) and side (right) views of a 3D reconstruction from the aligned cross-section FIB-SEM images show trajectories of cilia and the channel from the distal tip to the transition zone in WT and wsp-1a mutants. Colors: cilia. Grey: highly electron-dense material. Green: channel. Scale bar, 1 μm. (H) Longitudinal section TEM micrographs of the amphids in WT and wsp-1a mutant animals. Pink: socket cells. Blue: sheath cells. Brown: cilia. Yellow arrowheads: the collapsed socket cell. Yellow asterisks: highly electron-dense materials. Green asterisk: curved cilium. Scale bar, 1 μm. (I) Side views of a 3D reconstruction from the aligned longitudinal section FIB-SEM images show trajectories of cilia and the socket cells in WT and wsp-1a mutants. Colors: cilia. Pink: socket cells. Arrow: curved and branched cilia. Scale bar, 2 μm. (J) Bottom views of a 3D reconstruction from the aligned longitudinal section FIB-SEM images show the socket cell morphology in WT and wsp-1a mutant animals. Scale bar, 2 μm. (K) 3D reconstruction image shows Y-shaped cilia in wsp-1a mutant animals. (L) The axonemal MT structure of the ciliary middle or distal segments. Red arrowhead: doublet MTs. Scale bar, 100 nm. More images are shown in Supplementary information, Figure S1C. (M) Schematics of the axonemal MTs in WT and wsp-1a mutant cilia. (N-O) Localization of endogenous WSP-1A (N) and ARX-2 (O) around the amphid and phasmid of GFP knock-in strains. Cilia were labeled with OSM-6::mCherry (red). Arrowheads indicate the boundaries of the ciliary distal and middle segments. (P) Quantifications of the Dyf defect in the wsp-1a mutant and rescued animals. Two independent transgenic lines (L1 and L2) that express wsp-1a in the socket cells rescued Dyf defect in wsp-1a mutants. Error bars indicate SD (n = 350-400). (Q) Left: schematic of the phasmid socket cells (PHso1 and PHso2). Right: fluorescence images of F-actin (mCherry::utCH) in the phasmid socket cells of WT and wsp-1a mutant animals. (R) Quantifications of the length of F-actin fluorescence distribution in Q. Error bars indicate SD (n = 20-31). Scale bars in N, O, and Q, 2 μm. **P < 0.01, ***P < 0.001 based on Student's t-test; n.s., not significant.