Fig 2: Specific localization of ANKS1A to FOP-positive SDAs.a LWs (marked by dotted lines) with other forebrain tissues were subjected to X-gal staining. Scale bar, 500 µm. Cx cortex, CPu caudate putamen, ac anterior commissure. b The LWs were co-stained with ANKS1A, FOP, and CNTRL antibodies. The 3D SIM images were analyzed by the Imaris software. c 3DSIM micrographs of representative BBs. ANKS1A-VN was injected into the LV at E16.5 and subjected to in utero electroporation. The LWs at P4 were then co-stained for FOP and various BB markers with antibodies. Scale bar for b, c, 500 nm. d A median line and upper and lower quartile are presented in box and whisker plot of the axial distances of BB proteins from immature E1 cells. CEP164, n = 24; Centrin, n = 19; CNTRL, n = 14; ODF2, n = 15; ANKS1A-VN, n = 18. A green-shaded box marks a FOP-stained region brighter than 30% of the maximum intensity. e Table showing the radial distances of BB proteins from immature cells. The center of the FOP-positive ring image was used as a reference for the radial distances of BB proteins; n indicates the number of BBs used for statistical analysis. SD standard deviation. f Cartoon depiction of the BB proteins shown in d, e. g Whole-cell lysates (WCL) were prepared from six LWs at P4 and then the protein complexes were precipitated with the C-terminal specific antibody to detect the ANKS1A-associated proteins. h–j 3DSIM micrographs of BBs in E1 cells representing three different developmental stages. White arrowheads mark ANKS1A-VN present in FOP-stained SDAs. Scale bar for h–j, 500 nm. k Data shown in h–j were quantified with the total number of the double-positive FOP+CNTRL+ BBs set to 100%. Then the percentage of triple-positive ANKS1A+FOP+CNTRL+ BBs was calculated. Undifferentiated (UD), n = 67; immature (IM), n = 195; mature (M), n = 227 where n indicates the number of BBs analyzed.

Fig 3: ANKS1A loss results in partially impaired SDAs.a Representative 3DSIM images showing the gradual transition of SDAs from immature to the mature stage. Scale bar, 100 nm. b Cartoon depiction elucidating the dynamic change of SDAs from an unpolarized to a polarized state. c 3DSIM images were obtained from the anterior–dorsal (AD) region of LWs, and the BB patches were then magnified for further analysis. Schematic beside each panel is for the ×3.5 magnified white box and depicts the central angle of the FOP-negative region. Scale bar, 500 nm. d Data in c were quantified. Data are depicted as mean ± SD. Each point on the graph represents the central angle of the FOP-negative region. For the ANKS1A+/+ group, n = 356 BBs from 3 mice; for ANKS1A-/-, n = 412 BBs from 3 mice. e Experiments were performed as described in c, except that CNTRL and ODF antibodies were used. The boundary of each BB patch is outlined in white. Scale bar, 2 µm. f Data in e were quantified. Data are presented as mean ± SD. Each point on the graph represents the relative level of ODF2 intensity per BB patch. For the ANKS1A+/+ group, n = 68 cells from 2 mice; for ANKS1A-/-, n = 96 cells from 3 mice. g Number of BBs per cell was quantified based on their CNTRL staining, as illustrated in Supplementary Fig. 2c. Data represent mean ± SD. For the ANKS1A+/+ set, n = 81 cells from 3 mice; for ANKS1A-/-, n = 77 cells from 3 mice. h A ribbon of consecutive sections (50 nm) was placed on a one-hole grid and subsequently analyzed by TEM. Scale bar, 100 nm. i Schematic for clarifying the indices that represent the structural morphology of each BF. j, k The generated micrographs were aligned and used to measure the indices described in i. Data represent mean ± SD. For the ANKS1A+/+ group, n = 51 BBs; for ANKS1A-/-, n = 54 BBs.

Fig 4: ANKS1A loss results in severe SDA degeneration in aged mice.a Confocal microscopic analysis of LWs from littermates at 22 months of age (top panels). A representative cell (marked by yellow arrowheads) in each panel is enlarged to reveal motile cilia (green), BBs (red) (middle panels), and F-actin (gray) at the apical and subapical levels (bottom panels). The cell border and actin network around a BB patch are outlined with white and red dotted lines, respectively. b An SEM analysis of LWs from littermates at 22 months of age. Scale bar for a, b, 10 µm. c–e Data from a were quantified. The mice used for this quantification ranged from 18 to 22 months of age. For c: ANKS1A+/+ set, n = 110 cells from 4 mice; ANKS1A-/-, n = 113 cells from 4 mice. For d, e: ANKS1A+/+ group, n = 87 cells from 4 mice; ANKS1A-/-, n = 85 cells from 4 mice. f 3DSIM micrographs of LWs depict triple staining with FOP, ODF2, and CNTRL. White boxes indicate the regions of ×3 magnification that reveal SDA staining with each antibody (second and third panels). Scale bar, 10 µm. g Representative SDA images (top) together with their cartoon depiction (bottom). Scale bar, 200 nm. h The central angles were calculated. ANKS1A+/+ set, n = 164 BBs from 3 mice; ANKS1A-/-, n = 160 BBs from 3 mice. i Data in f were quantified. Data represent mean ± SD. ANKS1A+/+ group, n = 50 cells; ANKS1A-/-, n = 56 cells. j, k A ribbon of consecutive sections (50 nm) was placed on a one-hole grid and subsequently analyzed by TEM. The resulting micrographs were aligned to measure the overall BF size. Data represent mean ± SD. ANKS1A+/+ set, n = 50 BBs; ANKS1A-/-, n = 54 BBs. Scale bar, 100 nm.