Extended Data Fig. 8: Primary ciliary dysfunction resulted in astrocytic pruning and neuronal spine defects.

(a) Illustration of astrocytic engulfment analysis. (b-c) IMARIS volumetric reconstructions of astrocytes (green) containing Vglut1 (magenta) in the hippocampus in male control and Arl13b^cKO brains. (b’, c’) Enlarged region of (b) and (c) indicated by red squares. (b’, c’) cross-section images from the yellow line indicated locations in (b) and (c). Scale bars in (a-c), 5 µm. (d) Quantifications of the Vglut1⁺ puncta within astrocytes in the hippocampus of control (n = 6 astrocytes) and Arl13b^cKO (n = 9 astrocytes) from 3 male mice per group. (e) Quantifications of the Vglut1⁺ puncta within astrocytes in the upper (control, n = 17; Arl13b^cKO, n = 18 astrocytes) and deeper (control, n = 16; Arl13b^cKO, n = 16 astrocytes) layers of cortex, and hippocampus (control, n = 13; Arl13b^cKO, 17 astrocytes) from 3 female mice per group. (f, g) Quantifications of the Vglut1⁺ puncta engulfed by upper- (control, n = 19; Ift88^cKO, n = 20; Arl13b^cKO, n = 21; SmoM2-Arl13b^cKO, n = 20; mArl13b_rescue-Arl13b^cKO, n = 19; mArl13b^V358A_rescue-Arl13b^cKO, n = 14 astrocytes) and deeper- cortical astrocytes (control, n = 18; Ift88^cKO, n = 18; Arl13b^cKO, n = 18; SmoM2-Arl13b^cKO, n = 20; mArl13b_rescue-Arl13b^cKO, n = 14; mArl13b^V358A_rescue-Arl13b^cKO, n = 17 astrocytes) from 3 male mice per group. Arl13b^cKO mice in the mArl13b^WT or mArl13b^V358A groups were injected with AAV5-CAG-DIO-Arl13b^WT-mCh or AAV5-CAG-DIO-Arl13b^V358A-mCh viruses, respectively, together with AAV5-GFAP-GFP viruses, at P1. All mice were injected with tamoxifen from P7 to P9 and brains were collected at P35 for analysis. Vglut1⁺ puncta engulfed by astrocytes were analyzed by IMARIS. (h, i) Quantifications of spine volume, area, length, diameter in apical (control, n = 18 dendrites; Arl13b^cKO, n = 13 dendrites) and basal (control, n = 18 dendrites; Arl13b^cKO, n = 22 dendrites) dendrites from 3 male mice per group. (j, k) Quantifications of spine characteristics of M1 apical and basal dendrites from female mice. (l, m) Fraction of spine types in M1 cortex of female control and Arl13b^cKO mice. Data shown in (j-m) were analyzed from apical (control, n = 31 dendrites; Arl13b^cKO, n = 23 dendrites) and basal (control, n = 33 dendrites; Arl13b^cKO, n = 21 dendrites) dendrites from 3 female mice per group. Comparisons of two groups were performed using unpaired two-tailed Student’s t-test and comparisons of six groups were performed using one-way ANOVA followed by Tukey’s test. *P < 0.05, **P < 0.01, ***P < 0.001.