A novel Agbl5 mutant mouse model exhibited domed head and reduced lifespan.

(A) Schematic representation of the knock-out/knock-in strategy to create the novel Agbl5 mutant (Agbl5M1) allele. A region between start codon and intron 8 was replaced with a tdTomato reporter cassette using CRISPR/CAS9 system. (B) RT-PCR using primers targeting a region spanning exon 6 and exon 8 of Agbl5 confirmed the absence of Agbl5 transcripts in brain, eye, spinal cord, spleen, and testis of Agbl5M1/M1 mice. NC, negative control. (C) The Kaplan-Meier survival curve showed that the Agbl5M1/M1 mice hardly survived more than 50 days (p<0.0005, log-rank (Mantel-Cox) test). (D-E’) Representative images of P50 wild-type (D, D’) and Agbl5M1/M1 (E, E’) mice. Different from the wild-type mice (D, D’), the mutant (E, E’) developed dome-shaped head as indicated with red lines (D’, E’).

Histological and flow assessment of the cerebrospinal fluid (CSF) pathway.

Hematoxylin-Eosin staining on a serial coronal brain sections of P46 wild-type (WT, A-C) and Agbl5M1/M1 (D-F) mice. Markedly enlarged lateral ventricles (LV, D, closed arrowhead), the third ventricles (D, open arrowheads, D’), and the fourth ventricle (F) were observed in Agbl5M1/M1 mice compared to the corresponding part in wild-type mice (A, B and C, arrowheads), while the size of the aqueduct was less affected in the mutant (B vs. E, arrowheads). A’ and D’ are the close view of ventral 3rd ventricles that were indicated in dashed boxes in A and D respectively. The dorsal and ventral 3rd ventricles are pointed with open arrowheads in red and purple respectively in A and D. (G-N) Flow assessment of CSF in mice of P30 intraventricularly injected with Evans Blue. Whole brains were fixed in 4% PFA 20 min later after injection to allow ink distribution and diffusion in the brain of wild-type (G-J) and mutant mice (K-N). Tissue sections showed that diffused ink was clearly observed in the lateral ventricles (LV, red arrowheads in G, K), the third dorsal (d3V, red arrowheads in H, L) and ventral ventricles (v3V, purple arrowheads in H, L), the fourth ventricles (4thV, red arrowheads in J, N) and aqueducts (Aq, red arrowheads in I, M) of both wild-type (G-J) and mutant (K-N) mice, indicative of communicating hydrocephalus. Scale bars: 2 mm for A-N; 200 μm for A’, D’.

Aberrant ependymal multicilia in Agbl5M1/M1hydrocephalic mice.

(A-B) Representative images of the whole-mount lateral walls of LVs from P45 wild-type (WT, A) or Agbl5M1/M1 (B) immunostained with the ciliary marker, acetylated tubulin (Ac-Tub). While multicilia in WT ependyma evenly cover the ventricle surface and point to the same direction, mutlicilia bundles were scattered with many Act-Tub positive cilia lying on the cell surface. (C-F) Scanning electron microscopy analysis of LV walls from wild-type (C, E) and Agbl5M1/M1 (D, F) mice of P30. In the wild-type LV, ependymal cell were covered with evenly distributed cilia bundles in a uniformed direction (C), while in the mutant mice, cilia only in the middle of ependymal cell surface remain (D). (E, F) Higher magnification image showed that the length of remaining ependymal cilia in Agbl5M1/M1 mice (F) is similar to that in wild-type animal (E). (G) Sequential images of ciliary beating in wild-type (upper row) and Agbl5M1/M1 (lower row) mice. (H-I) Quantification of beating frequency of ependymal cilia (H) or the consistency of their beating directions (reflected by mean vector length) in individual cells (I) of P45 mice (from 3 wild-type and 2 mutant mice). Scale bar, A-D, 20 μm; E-F, 2μm, G, 5 μm.

The apical actin network in Agbl5M1/M1 ependymal cells is disrupted.

(A-L) immunostained with the centriolar distal appendage marker CEP164 (B, H) with actin network labeled with phalloidin (A, G, G, J). While BBs are clustered and polarized in the wild-type ependymal cells (B, C), those in the mutant (H, I) are often diffused. The actin networks are largely disrupted in the mutant ependymal cells G, J, vs. A, D for wild-type). (D-E’, J-K’) Z-projection views of apical actin network around BB in wild-type (C, E) and Agbl5M1/M1 (J, K) ependymal cells and respective orthogonal views (E’, K’). The mutant ependymal cells lack the compact actin networks even around clustered BBs. (F, L) Quantification of ependymal cells with differently distributed BBs in wild-type (F) and Agbl5M1/M1 mice (L). (M, N) Quantification of the total intensity of F-actin around BBs (M) and the intensity of F-actin per BB (N). Scale bar, A-C, G-I-, 20 μm; D-E’, J-K’2 μm.

Expression of multiciliogenesis-promoting protein is not impaired in Agbl5M1/M1 ependyma.

(A-B’) Immunofluorescence analysis revealed that tdTomato signals can be detected in heterozygous Agbl5M1 (Agbl5WT/M1) brain (B, B’), but not in the wild-type control (A, A’). The tdTomato signals were localized in the ependymal cells but largely devoid from the subventricular zone (arrowhead). At the dorsal-lateral region of the LV, the tdTomato signals extend to 2-3 layers (arrow). (C-E) In brain section of P12 Agbl5WT/M1 mice, tdTomato expression is colocalized with that of S100β, an ependymal cell marker along the surface of lateral ventricles. (F, G) Lateral ventricles from P7 wild-type and Agbl5M1/M1 mic were immunostained with Foxj1, a protein promoting multiciliation. (H) Quantification showed that th number of Foxj1-positive cells per length of LV walls in the mutant mice (n=5) is decreased in the dorsal wall, but unchanged or slightly increased in the lateral and middle walls respectively compared to that in the wild-type mice (n=5). Error bars represent SEM. ***, p<0.001; student’s t-test. Scale bars, A, B, 75 μm; A’, B’, 25 μm; C-E,10 μm, F-G, 50 μm.

The glutamylation level is increased in ependymal multicilia of Agbl5M1/M1 mice.

(A) A schematic representation shows the enzymes involved in tubulin polyglutamylation and modification recognized by GT335 and polyE antibodies respectively. (B) Immunoblotting of LV from mice of different ages showed that compared to the wild-type, the immunosignals of GT335 but not that of polyE are increased in Agbl5M1/M1 mice at all ages examined. (C-J) Lateral ventricles of P7 wild-type (C-F) and Agbl5M1/M1 (G-J) mice stained with GT335 (green) and DAPI. Representative images show that the intensity and length of GT335 immunosignals in ependymal cilia are increased in all three (H, dorsal; I, lateral; J, middle) walls of LV in the mutant mice compared with the respective walls in wild-type LVs (D, dorsal; E, lateral; F, middle). (K) The number of multicilia tufts is comparable between the wild-type (n=3) and mutant mice (n=3) in the lateral walls. (L-O) LVs of wild-type (L, M) and Agbl5M1/M1 (N, O) mice immunostained for Arl13b (red) with nuclei visualized by DAPI staining. (R) Quantification showed that compared to that of the wild-type mice (n=3), the length of Arl13b signal in ependymal multicilia of Agbl5M1/M1mice (n=3) were not changed. (P-Q) LVs of wild-type (P) and Agbl5M1/M1(Q) mice co-immunostained for GT335 (red) and acetylated tubulin (Ac-Tub, green). (S,T) Quantification showed that compared to that of the wild-type mice (n=3), the length of ciliary GT335 signal (S) in ependymal multicilia of Agbl5M1/M1 mice (n=3) is significantly increased while that of Ac-Tub (T) is reduced. Letters in blue: D, dorsal wall; L, lateral wall, M, middle wall. Error bars represent SEM, student’s t-test. Scale bars, C, G, 100 μm; D-J, L-Q, 10 μm.

The initially formed ependymal multicilia in Agbl5M1/M1 mice are motile.

(A-B) Images of SiR-tubulin labeled whole-mount LVs from P15 wild-type (A) and Agbl5M1/M1 (B) mice show that ependymal multicilia are initially formed in the mutant. (C) Sequential images of ciliary beating of P15 wild-type (upper row) and Agbl5M1/M1 (lower row) showed that the multicilia of wild-type ependymal cells beat in similar direction, while that of mutant are asynchronously. White and yellow open arrowheads indicate respective beating directions of multicilia of two cells; the closed arrowhead points to multicilia of an individual cell beat in opposite directions. (D) Bundled Agbl5M1/M1 multicilia largely beat at the frequency slightly higher than that of wild-type (n=30 for each animal, 2 mice for each genotype). Error bars represent SD. (E) The consistency of cilia beating directions of WT and Agbl5M1/M1 ependymal cell in the tissue level reflected by the mean vector length of individual imaging field. Representative histograms of beating angles for each genotype, represented in polar coordinates. The area of each wedge is proportional to the percentage of angles in the corresponding angle range. (F, G) Whole-mount LVs from P15 wild-type (F) and Agbl5M1/M1 (G) mice were co-immunostained with Centrin (BB marker) and β-Catenin (cell boundary marker). (H, I) Traces of the intercellular junction labeled with β-Catenin of ependymal cells shown in F and G respectively. The purple arrows show the vectors drawn from the center of the apical surface to that of the BB patch. (J) Diagram showing the measurement of BB patch displacement. (K) Quantification showed that BB patches in Agbl5M1/M1 ependymal cells are not properly displaced (n= 387 from 5 wild-type mice; n=367 from 4 mutants), p<0.001, student’s t test. (L) Histogram of the distribution of BB patch angles in ependymal cells of WT (blue) and Agbl5M1/M1 (orange), (n=327 from 5 wild-type; n=233 from 4 mutants), p<0.001, Watson’s 2-sample U2 test. (M, N) Whole-mount lateral walls of LV from P10 WT (M) and Agbl5M1/M1(N) co-immunostained for β-Catenin and tyrosinated tubulin (Try-tub) showed that the polarization of MTs was not affected in ependymal cells of the mutant. (O, P) Quantification of ependymal cell apical surface delineated by β-Catenin staining showed that the mutant (N) exhibits larger area than that of wild-type (M), while the ratio of the area of BB patch over the area of apical surface is reduced in the mutant (n=189 from 4 wild-type and n=110 from 3 mutants). Scale bars, A-C, 5 μm; F, G, 20 μm.

Targeted disruption of CP domain alone in Agbl5 did not cause hydrocephalus, despite the increased glutamylation in ependymal cilia.

(A) Schematic representation of the knock-out/knock-in strategy to create a second Agbl5 mutant (Agbl5M2) allele that resembles the one used in previous studies (Wu et al., 2017). (B) RT-PCR using primers targeting deleted region in Agbl5M2 allele confirmed the absence of Agbl5 transcripts in brain, eye, and testis in Agbl5M2/M2 mice. NC, negative control. (C-D) Similar to that in Agbl5WT/M1 mice, tdTomato immunosignal is detected in ependymal cells of P7 AgblM2 heterogenous mice (D, arrows). (E-F) Hematoxylin-Eosin staining of coronal sections of brains from 3-month old wild-type (E) and Agbl5M2/M2 (F) mice, where no enlarged ventricles were observed. (G) Immunoblotting assay showed that the tubulin glutamylation level was increased in the brain of both Agbl5 mutants compared to that in the wild-type. (H-I) Immunostaining showed that the ciliary GT335 signals in ependymal cells of Agbl5M2/M2 mice (I) are increased compared with that of the wild-type (H). (J-K) The ciliary acetylated-tubulin (Ac-Tub) signals are reduced in ependymal cilia in Agbl5M2/M2 mic (K) compared with that in the wild-type (J). L, lateral wall. Scale bars, C, D, 25 μm; E, F, 500 μm; H-K, 10 μm.