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. Author manuscript; available in PMC: 2026 Aug 18.
Published in final edited form as: Sci Transl Med. 2021 Mar 31;13(587):eaay7896. doi: 10.1126/scitranslmed.aay7896

Dysfunction of the proteoglycan Tsukushi causes hydrocephalus through altered neurogenesis in the subventricular zone in mice

Naofumi Ito 1,2,*, M Asrafuzzaman Riyadh 1,2,3,*, Shah Adil Ishtiyaq Ahmad 1,2,4,*, Satoko Hattori 5, Yonehiro Kanemura 6, Hiroshi Kiyonari 7, Takaya Abe 7, Yasuhide Furuta 7,8, Yohei Shinmyo 1,2,9, Naoko Kaneko 10, Yuki Hirota 10,11, Giuseppe Lupo 12, Jun Hatakeyama 13, Athary Abdulhaleem M Felemban 1,2,14, Mohammad Badrul Anam 1,2,15, Masahiro Yamaguchi 16, Toru Takeo 17, Hirohide Takebayashi 18, Minoru Takebayashi 19, Yuichi Oike 20, Naomi Nakagata 17, Kenji Shimamura 13, Michael J Holtzman 21, Yoshiko Takahashi 22,23, Francois Guillemot 24, Tsuyoshi Miyakawa 5, Kazunobu Sawamoto 10,25, Kunimasa Ohta 1,2,15,23,26,†
PMCID: PMC13480463  NIHMSID: NIHMS2188018  PMID: 33790026

Abstract

The lateral ventricle (LV) is flanked by the subventricular zone (SVZ), a neural stem cell (NSC) niche rich in extrinsic growth factors regulating NSC maintenance, proliferation, and neuronal differentiation. Dysregulation of the SVZ niche causes LV expansion, a condition known as hydrocephalus; however, the underlying pathological mechanisms are unclear. We show that deficiency of the proteoglycan Tsukushi (TSK) in ependymal cells at the LV surface and in the cerebrospinal fluid results in hydrocephalus with neurodevelopmental disorder-like symptoms in mice. These symptoms are accompanied by altered differentiation and survival of the NSC lineage, disrupted ependymal structure, and dysregulated Wnt signaling. Multiple TSK variants found in patients with hydrocephalus exhibit reduced physiological activity in mice in vivo and in vitro. Administration of wild-type TSK protein or Wnt antagonists, but not of hydrocephalus-related TSK variants, in the LV of TSK knockout mice prevented hydrocephalus and preserved SVZ neurogenesis. These observations suggest that TSK plays a crucial role as a niche molecule modulating the fate of SVZ NSCs and point to TSK as a candidate for the diagnosis and therapy of hydrocephalus.

INTRODUCTION

Hydrocephalus is a neurological disorder affecting all ages from children to the elderly. The disease is characterized by the abnormal expansion of the lateral ventricle (LV) and accompanied by severe neurological symptoms. Despite the large number of patients and the severity of this condition, limited information is available regarding its etiology (1, 2). Hydrocephalus can be caused not only by an abnormal flow of the cerebrospinal fluid (CSF) or by obstruction of the ventricular system but also by an altered regulation of neural stem cell (NSC) proliferation and differentiation within the subventricular zone (SVZ) neurogenic niche (3). For example, Bardet-Biedl Syndrome, a mental and visual disorder often accompanied by hydrocephalus, is associated with both ciliary dysfunction and altered numbers of neural progenitors in mice (4). Mutations in the neural cell adhesion molecule L1CAM cause defects in dendritic arborization and axonal elongation and lead to hydrocephalus along with neurodevelopmental disorders (5, 6). Hydrocephalus can be accompanied by other neurodevelopmental disorders, such as autism spectrum disorders (ASD) and attention-deficit hyperactivity disorder (ADHD), which occur in childhood with high prevalence and lifelong effects (7–9). Because of the association of hydrocephalus with various neurological disorders, identifying the molecular mechanisms of this condition is of paramount importance for the diagnosis and therapy of a broad spectrum of neuropathologies (10). Furthermore, although the role of postnatal neurogenesis in the human brain remains controversial, understanding its regulatory mechanisms in different organisms may expand the therapeutic options for the treatment of neurological diseases (11–13).

We previously identified Tsukushi (TSK), a small leucine-rich proteoglycan family protein acting as a key coordinator of the extracellular biochemical milieu in several embryonic signaling centers (14), such as the gastrula organizer (15), the neural plate border (16), the eye ciliary margin (17), the hippocampus (18), and in the homeostasis of adult tissues/organs (19, 20). At the molecular level, TSK works by directly binding and modulating ligands or receptors of key signaling pathways, such as transforming growth factor–β and Wnt (15, 17), but its mechanism of action in different contexts is still largely unknown. Because NSC self-renewal and differentiation are controlled by Wnt signaling (21), we hypothesized that TSK might regulate mammalian neurogenesis upstream of the Wnt pathway. In this study, we show that TSK produced by the ependymal cells flanking the LV is critical for NSC regulation in the mouse SVZ through the modulation of Wnt signaling. By causing excessive neuronal differentiation and apoptosis in the SVZ niche, TSK dysfunction leads to hydrocephalus during postnatal stages and to neurological deficits in adult mice. TSK variants found in patients with hydrocephalus were unable to regulate Wnt signaling and SVZ neurogenesis and could not functionally replace wild-type (WT) TSK. These results provide insight on the etiology of hydrocephalus, which may pave the way toward more effective diagnostic and therapeutic approaches for this condition.

RESULTS

Loss of TSK function causes neurodevelopmental disease-like phenotypes in mice

To assess the impact of TSK inactivation on neurological development, we performed a detailed behavioral analysis of previously described TSK knockout (TSK-KO) mice (22). We observed no changes in body weight or body temperature in these mutants (fig. S1, A and B). TSK-KO mice retained their grip for an extended period, showing no alterations in grip strength, coordination exercises, and sensation (fig. S1, C to F). General activity did not differ between WT and TSK-KO mice (fig. S1, G to J).

In the light/dark transition test (Fig. 1, A to D), TSK-KO mice showed a reduction in the distance traveled (P = 0.0031) and in the stay time (P = 0.0016) in the light chamber and a decrease in the number of light/dark transitions (P = 0.0173). We detected no differences between WT and TSK-KO mice in the elevated plus maze (Fig. 1, E to H). In the social interaction test (Fig. 1, I to M), we observed an increase in the total contact duration (P = 0.0062) and the mean duration/contact (P = 0.0332) in new environments, whereas the total number of active contacts decreased (P = 0.0491). Social interaction in home cage did not differ between genotypes, although we detected a difference in the activity level during the initial phase (from day 1 19:00 to day 3 6:00; P = 0.0446) between WT and TSK-KO (fig. S1, K to N). We also performed the three-chamber social approach test in TSK-KO mice. In the sociability test, both TSK-KO and WT mice spent more time around the “stranger1 side” cage or chamber (in which a mouse is placed) than in the “empty side” cage or chamber (fig. S2, A to D). In the social novelty preference test, TSK-KO mice showed no difference in the time spent in chamber or around cages when comparing the “familiar side” and “stranger2 side” (fig. S2, E to H). There was no genotype effect on the time spent around cages (fig. S2, C and D and G and H).

Fig. 1. Behavior test battery and anxiety and walking analysis in TSK-KO mice.

Fig. 1.

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(A to D) Light/dark transition test (WT, N = 18; TSK-KO, N = 18). (A) Total distance traveled, P = 0.0031 (Light, Distance) and P = 0.1178 (Dark, Distance). (B) Time spent in the light chamber for the light-dark transition test. P = 0.0016. (C) Number of transitions between light and dark boxes. P = 0.0173 (D) Latency to light, P = 0.1142. (E to H) Elevated plus maze (WT, N = 17; TSK-KO, N = 18). (E) Total number of entries into open and closed arm. P = 0.1252. (F) Percentage of the number of entries into the open arms. P = 0.1728. (G) Total distance of traveled. P = 0.1557. (H) Percentage of time spent in the open arms. P = 0.1506. (I to M) Social interaction and novel environment [WT, N = 8; TSK-KO, N = 9, P = 0.0062 (I), 0.0854 (J), 0.0491 (K), 0.0332 (L), 0.7654 (M)]. (N and O) Startle response and prepulse inhibition [WT, N = 17; TSK-KO, N = 18, P = 0.1064 (N), 0.012 (O) (110 dB Startle), 0.0022 (O) (120 dB Startle)]. (P to S) Beam walking test. Wide condition [WT = trials 1 to 4, N = 15; TSK-KO, N = 16, P = 0.0404 (P), 0.0296 (Q), 0.0224 (R), 0.227 (S)] and narrow condition [TSK-KO = trials 1 to 6, N = 15; TSK-KO, N = 17, P = 0.1187 (P), 0.0203 (Q), 0.1309 (R), 0.0415 (S)]. Statistical significances are denoted by P value, using one-way ANOVA or two-way repeated measure ANOVA. NS = not significant (≥0.05), *P < 0.05, and **P < 0.01.

In the forced swim, the percentage of the immobility time and distance traveled were comparable for TSK-KO and WT mice (fig. S1, O and P). In the tail suspension test, there was no difference between WT and TSK-KO mice (fig. S1Q). In TSK-KO mice, prepulse inhibition increased compared to WT [P = 0.012 (O, 110 dB Startle) and 0.0022 (O, 120 dB Startle)], irrespective of the difference in sound pressure levels (Fig. 1, N and O). We assessed sensorimotor coordination and motor learning by the balance beam test, observing lower moving speed in the wide passage (P = 0.0404; Fig. 1P), slip numbers in the narrow and wide passages (P = 0.0296 and 0.0203; Fig. 1Q), average speed in the wide passage (P = 0.0224; Fig. 1R), and prolonged latency in the narrow passage (P = 0.0415; Fig. 1S) in TSK-KO compared to WT mice. To measure the effects of TSK deficiency in working memory, we conducted the T-maze test, detecting no difference between WT and TSK-KO mice (fig. S2I). For the assessment of spatial learning and memory, we performed Barnes maze test and observed no difference between WT and TSK-KO (fig. S2, J to P).

TSK-KO mice develop communicating hydrocephalus

Cranial enlargement and hydrocephalus are prominently observed in various neurodevelopmental diseases (23). A recent survey reported that childhood macrocephaly is a risk marker for neurodevelopmental disease (24, 25). We did not observe cranial enlargement in TSK-KO mice (Fig. 2A). Instead, TSK-KO mice at postnatal day 10 (P10) had a smaller brain compared to WT (brain length, P = 0.000012; brain width, P = 0.000018; Fig. 2, B and C), along with a marked ventricular expansion (Fig. 2, D to G), which became detectable at P1 (P = 0.003) and greatly increased during postnatal life (P = 0.00076 for 6-month-old mice). LV expansion often results from obstruction of the ventricular system or failure of CSF circulation (26); however, we could not detect changes in the structure of the aqueduct and subcommissural organ (which are associated with hydrocephalus) at P0 stage preceding LV expansion (Fig. 2, H to J). Injecting a dye into one hemisphere of P10 mouse brains, when TSK-KO LV is already expanded, showed no difference between genotypes in the mobility of the dye in the ventricular system (Fig. 2, K and L). The direction of fluid flow generated by the ependymal cilia (27) was not changed in TSK-KO brains (movie S1). In conclusion, TSK-KO mice displayed a patent LV expansion with no cranial enlargement, ventricular obstruction, or abnormal CSF flow, suggesting that TSK deficiency results in communicating hydrocephalus.

Fig. 2. Structural analysis of TSK-KO brain.

Fig. 2.

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(A) Appearance of TSK-KO mice. Dotted lines indicate shape of head. (B) Whole brain of TSK-KO mice at P10. Scale bars, 2 mm. (C) Brain length and width at P10 [WT, N = 8; TSK-KO, N = 11, P = 0.000012 (length) and P = 0.000018 (width)]. (D to F) Section of TSK-KO mice brain at P10. (D) Coronal section. Scale bars, 1 mm. (E) Sagittal section. Scale bars, 2 mm. (F) Horizontal section. Arrows indicate LV. Scale bars, 1 mm. (G) Size of the LV. [P0, WT, N = 10 and TSK-KO, N = 8; P1, WT, N = 13 and TSK-KO, N = 12; P2, WT, N = 8 and TSK-KO, N = 7; P7, WT, N = 3 and TSK-KO, N = 3; P10, WT, N = 3 and TSK-KO, N = 3; P = 0.307 (P0) 0.003 (P1), 0.002 (P2), 0.00002 (P10), 0.00085 (P30), and 0.00076 (6 months)]. (H) Coronal section of TSK-KO mice at P0. Third ventricle, V3; aqueduct, AQ. Scale bars, 1 mm. (I) The aqueduct at P0. Scale bars, 200 μm. (J) The subcommissural organ at P0. Scale bars, 50 μm. (K) CSF flow blockage assay. Image was obtained from 10 min after dye injection into P10 WT and TSK-KO mice brain. Arrows indicate injection points. (L) Quantification of (K), comparing the intensity of injected dye at cerebellum traveled through the ventricular system (WT, N = 4; TSK-KO, N = 3; P = 0.856). Statistical significances are denoted by P value. NS = not significant (≥0.05), **P < 0.01, and ***P < 0.001, using two-tailed unpaired Student’s t test.

TSK is expressed in ependymal cells of the mouse postnatal SVZ

The SVZ niche flanking the LV regulates NSC activity and CSF secretion and flow (28). 5-Bromo-4-chloro-3-indolyl- β-D-galactoside (X-gal) staining on brain sections of P10 TSK heterozygous mice [TSK-hetero, TSK+/β-geo, carrying a β-galactosidase (β-gal) reporter gene in one of the TSK alleles] revealed that TSK is expressed in the LV wall and in the choroid plexus (Fig. 3, A to C, and fig. S3A). Immunohistochemistry (IHC) for TSK, β-gal, and the NSC (B cell) marker glial fibrillary acidic protein or the ependymal (E) cell marker S100β indicated that TSK is expressed in E cells lining the LV (Fig. 3, D and E, and fig. S4, A and B). By Western blot, we detected TSK protein in the CSF (Fig. 3F and fig. S5). These data indicate that TSK is secreted from E cells and choroid plexus into the CSF.

Fig. 3. Expression of TSK in the postnatal mouse brain.

Fig. 3.

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(A) X-gal staining of TSK-hetero (TSK+/β-geo) and WT mice at P10. Scale bars, 1 mm. (B) Illustration of observation site. (C) Magnified X-gal staining image corresponding to the small squared regions shown in (B). (D and E) IHC of the LV in TSK-hetero (TSK+/β-geo) mice. Scale bars, 20 μm. (F) Western blotting of 10 μl of CSF (6.2 mg/ml) using anti-TSK antibody Mab1D3F1.

TSK expression in E cells is required for proper LV formation

To determine whether the LV expansion detected in TSK-KO mice depends on the lack of TSK expression in E cells, we generated conditional knockout (cKO) mice via recombination with Cre-loxP derivatives regulated by the following promoters: CAG (ubiquitous); Nestin and Sox2 (NSCs and intermediate neural progenitors); Emx1 (cerebral cortex and E cells); and FoxJ1 [E cells, rostral migratory stream (RMS), and olfactory bulb (OB)] (Fig. 4, A and B) (29–32). We observed LV expansion in CAG-Cre (P = 4.11 × 10−8), Emx1-Cre (P = 9.22 × 10−5), and FoxJ1-Cre cKO (P = 5.36 × 10−6) mice but not in Sox2-Cre and Nestin-Cre mice (Fig. 4, C and D). In addition, we generated TSK-KO mice with transgenic expression of TSK–green fluorescent protein (GFP) in tandem, regulated by each of the aforementioned Cre-dependent promoters (Fig. 4, E and F) and observed that the expanded LV phenotype of TSK-KO mice was rescued in all of these transgenic mice [WT versus TSK-KO (P = 2.52 × 10−5), TSK-KO versus TgCAG-TSK (P = 2.03 × 10−4), TSK-KO versus TgSox2-TSK (P = 1.28 × 10−5), TSK-KO versus TgNestin-TSK (P = 3.36 × 10−5), TSK-KO versus TgEmx-TSK (P = 2.14 × 10−5), and TSK-KO versus TgFoxJ1-TSK (P = 2.99 × 10−5)]. (Fig. 4, G and H). These results suggest that TSK expression around the LV is non-cell autonomously required for the correct morphogenesis of the LV.

Fig. 4. LV morphogenesis in TSK cKO and transgenic mice.

Fig. 4.

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(A) Generation of TSK-floxed mouse (TSKflox/flox) for conditional TSK-KO. Transcript of TSK is shown as box. Gray box indicate TSK protein coding regions. (B) Mating Emx-1Cre/+;TSK−/− and TSKflox/flox generated Emx1Cre/+;TSKflox/− as conditional TSK-KO mouse. (C) Coronal sections at LV region of different conditional TSK-KO via recombination in Cre driver and control mice at P10. (D) Size of the LV. P = 4.11 × 10−8 (pCAG-△TSK versus pCAGCre;/−;TSKflox/+), 0.823 (pScx2-△TSK versus pScx2Cre;/−;TSKflox/+), 0.559 (pNestin-△TSK versus pNestinCre;/−;TSKflox/+), 9.22 × 10−5 (pEmx-△TSK versus pEmxCre;/−;TSKflox/+), and 5.36 × 10−6 (pFoxJ1-△TSK versus pFoxJ1Cre;/−;TSKflox/+). Scale bars, 1 mm. (E) Scheme of the Z/TSK transgene for the generation of ectopic expression of TSK mice (Emx1Cre/+;Z/TSK hetero;TSK−/−). (F) Generation of ectopic expression mice on the TSK-KO background. Crossing Emx1Cre/+;TSK−/− with Z/TSK homo;TSK−/− mice results Emx1Cre/+;Z/TSK hetero;TSK−/− (Tg Emx1-TSK). Emx1+/+;Z/TSK hetero;TSK−/− was used as control of TSK-KO mice. (G and H) IHC with anti-GFP antibody with 3,3′-Diaminobenzidine (DAB) development for ectopic TSK expressing mice (G) and corresponding LV measurement (H). TSK-KOTg is control for ectopic expression. Scale bars, 1 mm. P = 2.52 × 10−5 (WT versus TSK-KO), P = 2.03 × 10−4 (TSK-KO versus TgCAG-TSK), P = 1.28 × 10−5 (TSK-KO versus TgSox2-TSK), P = 3.36 × 10−5 (TSK-KO versus TgNestin-TSK), P = 2.14 × 10−5 (TSK-KO versus TgEmx-TSK), and P = 2.99 × 10−5 (TSK-KO versus TgFoxJ1-TSK). Statistical significances are denoted by P value. NS = not significant (≥0.05) and ***P < 0.001, using two-tailed unpaired Student’s t test.

Loss of TSK function alters SVZ neurogenesis

NSCs are the source of both astroglia and neurons in the SVZ (33–35). During SVZ neurogenesis, NSCs (B cells) give rise to transit amplifying cells (C cells). C cells generate neuroblasts (A cells), which migrate along the RMS to the OB, where they undergo neuronal differentiation (36). To investigate whether neurogenesis is affected in the TSK-deficient SVZ, we performed IHC to compare the percentage of SVZ cells positive for Sox2 (a marker of both B and C cells and of other astrocyte cell types) and Nestin (B and C cell marker) in TSK-KO and WT mice (37, 38). This analysis showed an increase of Nestin+ (lateral SVZ, P = 0.0138; ventral SVZ, P = 0.015) and Sox2+/Nestin+ (lateral SVZ, P = 0.020; ventral SVZ, P = 0.021) cells, both in the lateral and in the ventral areas of the TSK-KO mice SVZ, although the total Sox2+ population was reduced (lateral SVZ, P = 0.0002; ventral SVZ, P = 0.012) (Fig. 5, A and B). Furthermore, labeling of B and C cells and their progeny by recombination with Nestin-GFP mice showed an excess of GFP+ cells in the dorsolateral LV wall of TSK-KO mice (Fig. 5C). To investigate whether these changes were due to altered cell proliferation, we injected WT and TSK-KO mice at P10 with 5-ethynyl-2′-deoxyuridine (EdU) and euthanized them after 6 hours. The number of EdU+ cells was similar in the WT and TSK-KO SVZ, although an increase was detected in the OB of mice lacking TSK (P = 0.0189) (Fig. 5, D to F). Although cell proliferation in the SVZ of TSK-KO mice was not affected at P10 (as assessed by EdU incorporation), we detected an increase at P0 by IHC for the proliferating cell marker Ki67, which revealed increased numbers of Ki67+ (lateral SVZ, P = 0.006; ventral SVZ, P = 0.027) and of Sox2+/Ki67+ (lateral SVZ, P = 0.010; ventral SVZ, P = 0.015) cells at this stage (Fig. 5, G and H). Last, we analyzed apoptosis using IHC for cleaved caspase-3 and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Both of these analyses showed an increase of apoptotic cells in the TSK-KO SVZ at P10 (caspase-3, P = 0.0161; TUNEL, P = 0.047) (Fig. 5, I to L, and fig. S3, B to F). Together, these results suggest that, in TSK-KO mice, an early increase in neural stem/progenitor cell proliferation in the newborn SVZ is followed by excessive apoptosis at P10, thus altering the regulation of the SVZ niche during the critical window in which abnormal LV expansion takes place in these mice.

Fig. 5. TSK inactivation affects the activity of the SVZ niche.

Fig. 5.

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(A) Expression of the NSC markers in P10 mice at SVZ. Scale bars, 20 μm. (B) Counts of NSC marker expressing cells. P = 0.0002 (lateral, Sox2+), 0.0138 (lateral, Nestin+), 0.020 (lateral, Sox2+ Nestin+), 0.012 (ventral, Sox2+), 0.015 (ventral, Nestin+), and 0.021 (ventral, Sox2+ Nestin+). (C) Nestin-GFP expression in WT and TSK-KO mouse brains. Scale bars, 1 mm. (D to F) IHC with anti-EdU. (D) SVZ. (E) OB. (F) Counts of the EdU-positive cells (WT, N = 3; TSK-KO, N = 3). P = 0.6602 (lateral), 0.0720 (ventral), and 0.0189 (OB). Scale bars, 20 μm. (G and H) IHC with anti-Sox2 and anti-Ki67 at P0 LV. (G) Representative confocal images. Scale bars, 20 μm. (H) Counts of the Ki67+ and Sox2+ cells at P0 (WT, N = 3; TSK-KO, N = 3). P = 0.006 (lateral, Ki67), 0.027 (ventral, Ki67), 0.010 (lateral, Ki67+ Sox2+), and 0.015 (ventral, Ki67+ Sox2+). (I and J) IHC of cleaved caspase-3 at P10 LV. (I) Representative confocal images. Scale bars, 20 μm. (J) Counts of cleaved caspase-3+ cells in the LV at P10 (WT, N = 3; TSK-KO, N = 3). P = 0.205 (lateral) and 0.0161 (ventral). (K and L) TUNEL assay at P10 mice brain LV. (K) Representative confocal images. Scale bars, 20 μm. (L) Counts of TUNEL+ cells in the LV at P10 (WT, N = 3; TSK-KO, N = 3). P = 0.047 (lateral) and 0.230 (ventral). Statistical significances are denoted by P value. NS = not significant (≥0.05), *P < 0.05, **P < 0.01, and ***P < 0.001, using two-tailed unpaired Student’s t test.

Aminoacidic changes found in patients with hydrocephalus abrogate TSK activity in mutant mice and result in hydrocephalus

To investigate whether TSK dysfunction is associated to patients with hydrocephalus, we performed TSK amplicon sequencing of combined genomic DNA isolated from 13 Japanese patients with congenital hydrocephalus and identified four missense variants in the TSK coding region (table S1). By checking TSK sequence in individual patients with hydrocephalus, we found two patients with quadruple variants, harboring R208C, S248N, V308I, and D344E amino acid changes in the TSK gene (table S2). To exclude the possibility of aneuploidy, we quantified the copy number of TSK sequence in these patients and found no chromosomal copy number abnormalities (fig. S6A). These quadruple variants are predicted to alter the three-dimensional structure of the TSK protein compared to its consensus sequence (fig. S6B).

Human and mouse TSK sequences share 85% protein identity (fig. S6C). We constructed variants of mouse TSK each carrying single point mutations corresponding to the human patient–derived variants (R209C, S249N, and V309I), plus a linkage-type mutated TSK (lmTSK) that contains R209C, S249N, and V309I mutations mentioned above. The D344E change detected in human patients could not be reproduced in mouse TSK, because the murine protein already has a Glu (E) in the corresponding position. The lmTSK reactivity toward the anti- TSK antibody Mab1D3F1 remained unchanged (fig. S6D). We next generated mice harboring a mutant TSK transgene that contains the three hydrocephalus-related aminoacidic changes described above in a TSK-KO background (TSKlmTSK/−) (Fig. 6, A and B). By mating TSKlmTSK/− with FoxJ1-Cre mice, lmTSK was specifically expressed in E cells driven by the FoxJ1 promoter. FoxJ1-dependent expression of lmTSK in TSK-KO mice, unlike that of WT-TSK, resulted in an expanded LV (P = 0.0016) (Fig. 6, C to F), indicating that the hydrocephalus-associated mutations abrogate TSK function in the context of LV formation.

Fig. 6. Functional analysis of TSK variants found in patients with hydrocephalus.

Fig. 6.

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(A) Generation of human TSK variant-mimicking mouse. SA, adenovirus splicing acceptor; STOP, PGK-Neo-SV40tpA; bpA, bovine growth hormone polyadenylation site. (B) Mating for obtaining conditional lmTSK expressing mice. (C to E) lmTSK knock-in mouse brain at P10. (C) FoxJ1Cre/+; TSKlmTSK/−. (D) FoxJ1Cre/+; TSKlmTSK/+. (E) TSK-hetero. Control for (C) and (D). Scale bars, 1 mm. (F) Size of the LV. P = 0.0016 (FoxJ1Cre/+; TSKlmTSK/− versus TSK-hetero) and 0.822 (FoxJ1Cre/+; TSKlmTSK/+ versus TSK-hetero). Statistical significances are denoted by P value. NS = not significant (≥0.05), **P < 0.01, using two-tailed unpaired Student’s t test.

TSK modulates Wnt signaling in the SVZ

To investigate the molecular mechanism underlying TSK-dependent regulation of LV morphogenesis, we analyzed the transcriptomic changes caused by TSK deficiency by performing RNA sequencing (RNA-seq) of SVZ tissue dissected from WT and TSK-KO mice (table S3). This analysis showed a strong up-regulation of several Wnt pathway–related genes in TSK-KO samples (table S4). Transmembrane Frizzled (Fzd) proteins regulate embryonic development and tissue homeostasis by acting as receptors for Wnt proteins and other ligands (39, 40). Wnt1 is the most abundant among Wnt molecules in the perinatal mouse brain (fig. S7, A and B) (41). Fzd3 is a major Wnt receptor in the brain (42). Its deletion affects several major axon tracts within the forebrain and results in LV expansion (43). We detected Fzd3 expression in neurospheres derived from the SVZ of both WT and TSK-KO mice [P = 0.0082 (Wnt2b-WT versus Fzd3-WT), P = 0.0076 (Wnt2b-WT versus Fzd4-WT), P = 0.0136 (Wnt2b-WT versus Fzd7-WT), P = 0.0143 (Wnt2b-TSK-KO versus Fzd3-TSK-KO), P = 0.0017 (Wnt2b-TSK-KO versus Fzd4-TSK-KO), and P = 0.0234 (Wnt2b-TSK-KO versus Fzd7-TSK-KO)]. (Fig. 7A). We found that Fzd3 binds TSK with high affinity in vitro [Kd (dissociation constant) = 2.7 × 10−10 M] (Fig. 7B), suggesting that TSK modulates Wnt signaling in the SVZ. To assess Wnt pathway involvement in TSK function, we injected TSK-KO pups with multiple doses of the small-molecule Wnt inhibitor WNT-C59 and observed the effect at P10. Wnt-C59 suppress WNT–β-catenin signaling expression and cellular proliferation in mouse mammary tumor virus WNT1 transgenic mice (44). Supporting our hypothesis, neonatal injection of WNT-C59 prevented the phenotypic LV expansion in TSK-KO mice [P = 0.017 (C59+WT versus C59+ TSK-KO), P = 0.000076 (C59+WT versus C59−TSK-KO), P = 0.029 (C59+ TSK-KO versus C59−TSK-KO), and P = 0.017 (C59−WT versus C59−TSK-KO)] (Fig. 7, C and D). Injection of WNT-C59 also increased the number of Sox2+ cells in all cases, which correlates with the trend shown in Fig. 5B, although the data were not statistically significant (Fig. 7, E to G). These data suggest that TSK regulates LV morpho genesis through the inhibition of Wnt signaling.

Fig. 7. Wnt1-Fzd3 signaling in TSK-KO mice.

Fig. 7.

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(A) Semiquantitative reverse transcription polymerase chain reaction analysis using neurosphere RNA. [P = 0.2943 (Wnt2b-WT versus TSK-WT), P = 0.0082 (Wnt2b-WT versus Fzd3-WT), P = 0.0076 (Wnt2b-WT versus Fzd4-WT), P = 0.0136 (Wnt2b-WT versus Fzd7-WT), P = 0.3369 (Wnt2b-TSK-KO versus TSK-TSK-KO), P = 0.0143 (Wnt2b-TSK-KO versus Fzd3-TSK-KO), P = 0.0017 (Wnt2b-TSK-KO versus Fzd4-TSK-KO), and P = 0.0234 (Wnt2b-TSK-KO versus Fzd7-TSK-KO)]. (B) Alkaline Phosphatase (AP) binding assays for ligand TSK and receptor Fzd3. (C) Coronal section of P10 mice brain that was injected WNT-C59 in 30% propylene glycol at P0 to P2. Thirty percent propylene glycol without WNT-C59 was injected as control. Scale bars, 1 mm. (D) Quantification of the LV area in WNT-C59–injected mice. (N; Wnt-C59+_WT = 10, WNT-C59+_TSK-KO = 6, Wnt-C59−_WT = 5, and Wnt-C59−_TSK-KO = 7). P = 0.017 (C59+WT versus C59+TSK-KO), P = 0.051 (C59+WT versus C59−WT), P = 0.000076 (C59+WT versus C59−TSK-KO), P = 0.029 (C59+TSK-KO versus C59−TSK-KO), and P = 0.017 (C59−WT versus C59−TSK-KO). (E to G) Sox2 IHC of WNT-C59–injected mice LV at P10. (E and F) Representative confocal images. Scale bars, 20 μm. (G) Ratio of LV flanked Sox2+ cells. (N; Wnt-C59+_WT = 3, Wnt-C59+_TSK-KO = 5, Wnt-C59−_WT = 3, and Wnt-C59−_TSK-KO = 3). P = 0.3072 (WT lateral, C59+ versus C59−), P = 0.5773 (WT ventral, C59+ versus C59−), P = 0.1608 (TSK-KO lateral, C59+ versus C59−), and P = 0.2029 (TSK-KO ventral, C59+ versus C59−). Statistical significances are denoted by P value. NS = not significant (≥0.05), *P < 0.05, **P < 0.01, and ***P < 0.001, using two-tailed unpaired Student’s t test.

TSK deficiency affects ciliated cells in the wall of the LV

To identify TSK-dependent gene expression networks associated with hydrocephalus, gene categories enriched among the differentially expressed genes between WT and TSK-KO SVZ were investigated with the Database for Annotation, Visualization and Integrated Discovery (DAVID) Gene Ontology (GO) database using the summarizing program Reduce + Visual Gene Ontology (REVIGO) (45, 46). This analysis revealed an enrichment for categories related to cilium assembly and astrocyte development in TSK-KO mice (table S5). Abnormalities in tight junctions and cilia may disrupt the absorption and flow of the CSF, resulting in hydrocephalus (47, 48). No abnormalities of ciliary polarity were observed along the LV surface (fig. S8, A to C), although electron microscopic analysis of the LV wall indicated that ciliated cells were more scattered in TSK-KO mice than in WT (fig. S8D). These results suggest that loss of TSK functions leads to alterations of ciliogenesis in the LV wall, although they are less severe than those detected in ciliogenesis-deficient mice (49).

Intraventricular injection of WT-TSK protein, but not of hydrocephalus-associated mutant TSK, prevents the hydrocephalic phenotype of TSK-KO mice

To study the biological activity of the TSK variants identified in human patients with hydrocephalus, we performed in vitro binding assays with Fzd3, finding that, although mouse TSK variants carrying single mutations bound Fzd3, the binding was abolished in the lmTSK mutant (Fig. 8, A and B, and fig. S9). Moreover, intraventricular injection of WT-TSK protein prevented LV expansion in TSK-KO mice, but lmTSK protein lacked this biological activity [P = 0.020 (WT-TSK versus control) and P = 0.029 (lmTSK versus control)] (Fig. 8, C and D). This rescue was mainly detectable in the anterior region of the injected LV (Fig. 8, E and F). Immunostaining against His and V5 showed that the exogenous TSK protein was localized in limited areas of the injected LV wall, whereas no staining was observed in the control lateral LV [P = 0.0000257 (N; Inj LV = 3, Non-inj LV = 3)] (Fig. 8, G and H). Together with the previously reported anterior-high posterior-low expression gradient of Fzd3 (fig. S8E) (41), these results suggest that TSK regulates LV morphogenesis by modulating Fzd3-dependent signaling in the LV wall.

Fig. 8. Loss of Fzd3 binding in hydrocephalus-associated TSK variants.

Fig. 8.

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(A) Coprecipitation assay. Anti-His-tag (top) and anti–Flag-tag (middle and bottom). WB, Western Blot; OD, Optical Density. (B) Measurement of binding kinetics with an AP-tagged TSK protein (TSK-AP), AP-tagged lmTSK protein (lmTSK-AP), and AP protein (AP only) with Fzd3. (C) Rescue of LV expansion via injection of TSK protein into TSK-KO mouse brain. TSK protein was injected at P0 and rescue of LV expansion was confirmed at P1. Arrows, injection site; control, supernatant of native COS-7 culture. Scale bars, 1 mm. (D) Relative LV area at the protein injection side to the noninjection side. N; WT-TSK protein = 4, control = 3, and lmTSK = 8. P = 0.020 (WT-TSK versus control) and P = 0.029 (lmTSK versus control). (E to F) Effect of TSK for the suppression of LV expansion along the anterior-posterior axis. (E) Serial section of TSK protein–injected TSK-KO mouse brain LV. Scale bars, 1 mm. (F) Rescue percentage (injected area/noninjected area) of LV sections aligned from anterior to posterior. (N; I = 7, II = 8, III = 8, IV = 7, and V = 7). (G) IHC of TSK protein injected brain. Injected TSK protein was carrying His and V5 tags. Dashed squares indicate magnification area. Scale bars, 100 μm. (H) Quantification of (G), showing the localization of injected TSK protein on LV wall. P = 0.0000257 (N; inj LV = 3, non-inj LV = 3). Statistical significances are denoted by P value. *P < 0.05 and ***P < 0.001, using two-tailed unpaired Student’s t test.

DISCUSSION

On the basis of the results of this study, we proposed the model summarized in fig. S10. According to this model, E cell–derived TSK protein acts on E cells to promote ciliogenesis and on the underlying SVZ niche to regulate proliferation and survival of neural stem/progenitor cells. In the absence of TSK function, the neurogenic process is altered because of overproduction and apoptosis of neural stem/progenitor cells, and E cell ciliogenesis is affected, leading to abnormal LV expansion and hydrocephalus-related neurological phenotypes, such as increased anxiety-like behavior, impaired social behavior, prepulse inhibition, motor imbalance, and loss of coordination. TSK released by E cells acts non-cell autonomously on the SVZ niche, as shown by (i) alterations of cell proliferation and survival in the SVZ following conditional TSK-KO in E cells, (ii) the rescue of LV expansion and SVZ neurogenesis by transgenic expression of TSK in E cells of TSK-KO mice, or (iii) by injection of TSK protein into the LV. We describe recurrent missense single nucleotide variations in the TSK gene of human congenital patients with hydrocephalus, which result in amino acid changes altering the three-dimensional structure and the functional activity of TSK, as shown by (i) disrupted binding of mutant TSK to Fzd3, (ii) LV expansion in TSK-KO mice expressing mutagenized TSK in E cells, and (iii) the inability of mutant TSK protein to rescue the phenotype of TSK-KO mice after injection into the LV.

TSK-KO mice exhibited impaired social, cognate, and motor skills, activation of prepulse inhibition, and gait disorders, which are prominent characteristics of ASD mouse models (25, 50, 51). Furthermore, anxiety-like behavior, a feature of ADHD models (52), was also observed in TSK-KO mice. Therefore, the behavioral patterns shown by TSK-KO mice were, at least in part, similar to those found in patients with psychiatric disorders, such as ASD and ADHD, in agreement with the known association of hydrocephalus with these neurological disorders. Changes in activity, sociality, and spatial memory were not observed in TSK-KO mice, which are diverse in ADHD-like mice (53–55).

Albeit we observed abnormal density of ciliated cells in TSK-KO mice, the direction of CSF flow was unaffected, and no ventricular obstructions were found in the absence of TSK function. Although we cannot completely exclude the possibility that an abnormal CSF flow causes the LV expansion observed in TSK-KO mice, our data suggest that TSK dysfunction leads to communicating hydrocephalus by altering SVZ neurogenesis rather than CSF flow. More studies are needed to clarify the functional consequences of the defects in ciliogenesis of TSK-KO mice.

The postnatal SVZ niche bordering the LV contains neural stem/progenitor cells derived from embryonic radial glia (56) and supplies newly generated neurons to the OB (57, 58), supporting plasticity of olfaction-related neuronal circuits (59). Maintenance and differentiation of NSCs are regulated by the input of several signaling pathways, where Wnt pathway is a major contributor (21). Our results suggest that TSK is implicated in the regulation of postnatal neurogenesis by modulating Wnt signaling in the SVZ niche. The SVZ niche is also regulated by the choroid plexus secretome released in the CSF (60). The Wnt1 ligand and the Fzd3 receptor are strongly expressed in the perinatal mice brain (41), and Fzd3-deficient mice show brain morphological defects including LV expansion (42, 61). The choroid plexus secretome contains Wnt molecules and stem cell maintenance factors and regulates the SVZ niche milieu (60, 62). Otx2 is a transcription factor essential for choroid plexus development, which controls the expression of Sostdc1 (Wise) and Msx1, and is ectopically expressed in the absence of Wnt1 function (63, 64). Our transcriptomic analysis showed that the expression of Otx2 and other Wnt1-related genes was increased in TSK-KO mice, suggesting that Wnt signaling may be abnormally enhanced in the TSK-KO SVZ. In agreement with this hypothesis, the hydrocephalus phenotype of TSK-KO mice could be mitigated by injecting the small-molecule Wnt antagonist compound C59 in the mutant LV. On the basis of these observations, we speculate that TSK released by E cells in the CSF and working as an antagonist of the transmembrane receptor Fzd3 attenuates the signaling activity of the CSF secretome to regulate the SVZ niche and brain morphogenesis. Thus, TSK variants associated to patients with hydrocephalus are unable to bind Fzd3 and support proper LV morphogenesis, suggesting that Fzd3 binding is critical for TSK function in the regulation of LV formation. TSK is also expressed in the choroid plexus, suggesting that it may also act as part of the choroid plexus secretome. Although the results shown in this work show that TSK expression in E cells is both necessary and sufficient to support LV development, in future studies, it will be interesting to address the role of TSK expression in the choroid plexus.

In conclusion, we show that TSK acts as a crucial extracellular niche molecule in the SVZ, which is required to maintain the optimal cell proliferation and survival and to assist proper LV formation. TSK-KO mice recapitulate the anatomical and neurological traits of hydrocephalus, and TSK variants found in patients affected by this condition abrogate TSK function. Despite these interesting results, the present study has some limitations. In particular, although the hydrocephalus phenotype of TSK-KO mice is associated with changes to cell proliferation and cell death in the SVZ neurogenic niche, a causal relationship between altered neurogenesis and hydrocephalus in these mice will have to be demonstrated in future work. Furthermore, although we have identified the cell types in which TSK expression is required for proper LV formation, additional work with conditional mutant mice will be needed to define the temporal window in which TSK function is necessary. Nonetheless, the data and tools generated in this study may help to elucidate the pathogenic mechanisms of hydrocephalus and to develop new diagnostic and therapeutic approaches for the prevention and treatment of this disease.

MATERIALS AND METHODS

Study design

This study mainly consists of in vivo experiments using TSK-KO mice and transgenic mice either lacking or overexpressing TSK proteins at specific cells in the mouse brain. Behavior test battery confirmed that the loss of TSK resulted in hydrocephalus with neurodevelopmental disorder-like symptoms such as motor imbalance and an increased anxiety-like behavior. Detail phenotypic analysis of TSK-KO brains was performed with whole brains and serial sections on coronal, sagittal, and horizontal planes. Using immunostaining, ependymal cell was identified to be specifically expressing TSK at LV wall. Transgenic mice were prepared using ependymal cell or NSC-specific promoters to specifically omit TSK gene or express TSK gene on a TSK-KO background. TSK gene was sequenced from 13 patients with hydrocephalus, and three potential amino acid conversions were detected. Mutant mouse TSK protein was constructed carrying these three amino acid conversions, and transgenic mice expressing the mutant TSK protein on TSK-KO background were generated using CRISPR-Cas9 system. Transcriptome analysis of WT and TSK-KO SVZ was performed using RNA-seq. TSK protein was produced in COS-7 culture and injected into P0 ventricle to observe the rescue of KO phenotype at P1. To confirm the involvement of Wnt pathway, Wnt inhibitor molecule WNT-C59 was administered subcutaneously at P0 to P2, and rescue was observed at P10. Sample size was not predetermined, and samples were not blinded or randomized. More method details are available in the Supplementary Materials.

Quantification and statistical analysis

No statistical methods were used to predetermine sample size. No blinding and randomizing were carried out. Number of samples (N) and method for significance analysis [one-way analysis of variance (ANOVA), two-way ANOVA, and two-tailed unpaired Student’s t test] are described in the figure legends individually. Boxplot and linear approximation were depicted by R packages.

Supplementary Material

Direction of fluid flow generated by the ependymal cilia in WT mice and TSK-KO mice.

Movie S1. Direction of fluid flow generated by the ependymal cilia in WT mice and TSK-KO mice.

Download video file (14.8MB, mp4)
RNA-seq WT vs TSK-KO
Supplemental Material

Fig. S1. Behavioral test battery: Global behavior.

Fig. S2. Behavioral test battery: Three-chamber sociability and social novelty test (Crawley version).

Fig. S3. β-gal staining and TUNEL assay in RMS.

Fig. S4. IHC of the LV in TSK-hetero (TSK+/β-geo) mice.

Fig. S5. Relative expression of TSK protein in 10 μl of CSF (6.2 mg/ml) of WT and TSK-KO mice using anti-TSK antibody Mab1D3F1.

Fig. S6. Human TSK identified from patients with congenital hydrocephalus.

Fig. S7. Wnt family expression in mice.

Fig. S8. Cilia polarity.

Fig. S9. Coprecipitation assay.

Fig. S10. Summary of this study.

Table S1. Amplicon sequencing results of 11 single nucleotide variants derived from 13 patients by TSK, Fzd3, Fzd4, and BMP2B primers.

Table S2. Sanger sequencing of DNA samples from patients with hydrocephalus.

Table S3. Expression profiles of WT and TSK-KO mice isolated LV tissue by RNA-seq.

Table S4. The alterations of Wnt signaling genes in TSK-KO mice from comparative RNA-seq.

Table S5. GO analysis of comparative transcriptome at LV.

stm.sciencemag.org/cgi/content/full/13/587/eaay7896/DC1

Materials and Methods

Data file S1.

References (65–97)

Acknowledgments:

We thank G. Eguchi, H. Fujisawa, H. Nakamura, and T. Suda for critical reading of the manuscript; T. Iwasato for providing Emx1-cre mice; M. Yamasaki, A. Harada, K. Nishiyama, Y. Fujii, M. Ikeno, S. Ninomiya, S. Fukumura, T. Yamanaka, Y. Nakamura, H. Arai, A. Okumura, T. Ohya, and N. Okamoto for providing human patient DNA samples; and S. Fujimura, M. Kumamaru, I. Arif, M. Iimori, K. Yamada, A. Hamashima, and M. Takiguchi for technical assistance. We appreciate the Institute of Molecular Embryology and Genetics, Gene Technology Center, School of Medicine Core Laboratory for Medical Research and Education, and the Instrumental Analysis Center of Pharmacy at Kumamoto University for providing experimental support.

Funding:

This work was supported by grants from KAKENHI (22122009), Kumamoto University Advanced Research Project Stem Cell-Based Tissue Regeneration Research and Education Unit, Program for Leading Graduate Schools “HIGO Program” in Kumamoto University, and Japan Agency for Medical Research and Development. Behavioral battery test was carried out at Institute for Comprehensive Medical Science, Fujita Health University (Joint Usage/Research Center for Genes, Brain, and Behavior) and is supported by a Grant-in-Aid for Scientific Research on Innovative Areas “Platform of Advanced Animal Model Support” (16H06276) from the Ministry of Education, Science, Sports, and Culture of Japan.

Footnotes

Competing interests: The authors declare that they have no competing financial interests.

Data and materials availability:

All data associated with this study are present in the paper or the Supplementary Materials.

REFERENCES AND NOTES

  • 1.Kahle KT, Kulkarni AV, Limbrick DD Jr., Warf BC, Hydrocephalus in children. Lancet 387, 788–799 (2016). [DOI] [PubMed] [Google Scholar]
  • 2.McAllister JP, Getting to the Root of Hydrocephalus. Sci. Transl. Med. 3, 99fs4 (2011). [DOI] [PubMed] [Google Scholar]
  • 3.Ohata S, Álvarez-Buylla A, Planar organization of multiciliated ependymal (E1) cells in the brain ventricular epithelium. Trends Neurosci. 39, 543–551 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Carter CS, Vogel TW, Zhang Q, Seo S, Swiderski RE, Moninger TO, Cassell MD, Thedens DR, Keppler-Noreuil KM, Nopoulos P, Nishimura DY, Searby CC, Bugge K, Sheffield VC, Abnormal development of NG2+PDGFR-α+ neural progenitor cells leads to neonatal hydrocephalus in a ciliopathy mouse model. Nat. Med. 18, 1797–1804 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Okamoto N, Del Maestro R, Valero R, Monros E, Poo P, Kanemura Y, Yamasaki M, Hydrocephalus and Hirschsprung’s disease with a mutation of L1CAM. J. Hum. Genet. 49, 334–337 (2004). [DOI] [PubMed] [Google Scholar]
  • 6.Patzke C, Acuna C, Giam LR, Wernig M, Südhof TC, Conditional deletion of L1CAM in human neurons impairs both axonal and dendritic arborization and action potential generation. J. Exp. Med. 213, 499–515 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Xu G, Strathearn L, Liu B, Yang B, Bao W, Twenty-year trends in diagnosed attention-deficit/hyperactivity disorder among US Children and Adolescents, 1997–2016. JAMA Netw. Open 1, e181471 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Elsabbagh M, Divan G, Koh Y-J, Kim YS, Kauchali S, Marcín C, Montiel-Nava C, Patel V, Paula CS, Wang C, Yasamy MT, Fombonne E, Global prevalence of autism and other pervasive developmental disorders. Autism Res. 5, 160–179 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.American Psychiatric Association, Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association, Ed. 5, 2013). [Google Scholar]
  • 10.Thapar A, Cooper M, Rutter M, Neurodevelopmental disorders. Lancet Psychiatry 4, 339–346 (2017). [DOI] [PubMed] [Google Scholar]
  • 11.Hagihara H, Murano T, Ohira K, Miwa M, Nakamura K, Miyakawa T, Expression of progenitor cell/immature neuron markers does not present definitive evidence for adult neurogenesis. Mol. Brain 12, 108 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Moreno-Jiménez EP, Flor-García M, Terreros-Roncal J, Rábano A, Cafini F, Pallas-Bazarra N, Ávila J, Llorens-Martín M, Adult hippocampal neurogenesis is abundant in neurologically healthy subjects and drops sharply in patients with Alzheimer’s disease. Nat. Med. 25, 554–560 (2019). [DOI] [PubMed] [Google Scholar]
  • 13.Sorrells SF, Paredes MF, Cebrian-Silla A, Sandoval K, Qi D, Kelley KW, James D, Mayer S, Chang J, Auguste KI, Chang EF, Gutierrez AJ, Kriegstein AR, Mathern GW, Oldham MC, Huang EJ, Garcia-Verdugo JM, Yang Z, Alvarez-Buylla A, Human hippocampal neurogenesis drops sharply in children to undetectable levels in adults. Nature 555, 377–381 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ahmad SAI, Anam MB, Ito N, Ohta K, Involvement of Tsukushi in diverse developmental processes. J. Cell Commun. Signal 12, 205–210 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Ohta K, Lupo G, Kuriyama S, Keynes R, Holt CE, Harris WA, Tanaka H, Ohnuma S-I, Tsukushi functions as an organizer inducer by inhibition of BMP activity in cooperation with chordin. Dev. Cell 7, 347–358 (2004). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Kuriyama S, Lupo G, Ohta K, Ohnuma S-I, Harris WA, Tanaka H, Tsukushi controls ectodermal patterning and neural crest specification in Xenopus by direct regulation of BMP4 and X-delta-1 activity. Development 133, 75–88 (2006). [DOI] [PubMed] [Google Scholar]
  • 17.Ohta K, Ito A, Kuriyama S, Lupo G, Kosaka M, S.-i. Ohnuma, S. Nakagawa, H. Tanaka, Tsukushi functions as a Wnt signaling inhibitor by competing with Wnt2b for binding to transmembrane protein Frizzled4. Proc. Natl. Acad. Sci. U.S.A. 108, 14962–14967 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Ahmad SAI, Anam MB, Istiaq A, Ito N, Ohta K, Tsukushi is essential for proper maintenance and terminal differentiation of mouse hippocampal neural stem cells. Dev. Growth Differ. 62, 108–117 (2020). [DOI] [PubMed] [Google Scholar]
  • 19.Niimori D, Kawano R, Felemban A, Niimori-Kita K, Tanaka H, Ihn H, Ohta K, Tsukushi controls the hair cycle by regulating TGF-β1 signaling. Dev. Biol. 372, 81–87 (2012). [DOI] [PubMed] [Google Scholar]
  • 20.Wang Q, Sharma VP, Shen H, Xiao Y, Zhu Q, Xiong X, Guo L, Jiang L, Ohta K, Li S, Shi H, Rui L, Lin JD, The hepatokine Tsukushi gates energy expenditure via brown fat sympathetic innervation. Nat. Metab. 1, 251–260 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Kalani MYS, Cheshier SH, Cord BJ, Bababeygy SR, Vogel H, Weissman IL, Palmer TD, Nusse R, Wnt-mediated self-renewal of neural stem/progenitor cells. Proc. Natl. Acad. Sci. U.S.A. 105, 16970–16975 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Ito A, Shinmyo Y, Abe T, Oshima N, Tanaka H, Ohta K, Tsukushi is required for anterior commissure formation in mouse brain. Biochem. Biophys. Res. Commun. 402, 813–818 (2010). [DOI] [PubMed] [Google Scholar]
  • 23.Bolton PF, Roobol M, Allsopp L, Pickles A, Association between idiopathic infantile macrocephaly and autism spectrum disorders. Lancet 358, 726–727 (2001). [DOI] [PubMed] [Google Scholar]
  • 24.Kurata H, Shirai K, Saito Y, Okazaki T, Ohno K, Oguri M, Adachi K, Nanba E, Maegaki Y, Neurodevelopmental disorders in children with macrocephaly: A prevalence study and PTEN gene analysis. Brain Dev. 40, 36–41 (2018). [DOI] [PubMed] [Google Scholar]
  • 25.Bourgeron T, From the genetic architecture to synaptic plasticity in autism spectrum disorder. Nat. Rev. Neurosci. 16, 551–563 (2015). [DOI] [PubMed] [Google Scholar]
  • 26.Banizs B, Pike MM, Millican CL, Ferguson WB, Komlosi P, Sheetz J, Bell PD, Schwiebert EM, Yoder BK, Dysfunctional cilia lead to altered ependyma and choroid plexus function, and result in the formation of hydrocephalus. Development 132, 5329–5339 (2005). [DOI] [PubMed] [Google Scholar]
  • 27.Sawamoto K, Wichterle H, Gonzalez-Perez O, Cholfin JA, Yamada M, Spassky N, Murcia NS, Garcia-Verdugo JM, Marin O, Rubenstein JLR, Tessier-Lavigne M, Okano H, Alvarez-Buylla A, New neurons follow the flow of cerebrospinal fluid in the adult brain. Science 311, 629–632 (2006). [DOI] [PubMed] [Google Scholar]
  • 28.Fuentealba LC, Obernier K, Alvarez-Buylla A, Adult neural stem cells bridge their niche. Cell Stem Cell 10, 698–708 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Gorski JA, Talley T, Qiu M, Puelles L, Rubenstein JLR, Jones KR, Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-Expressing Lineage. J. Neurosci. 22, 6309–6314 (2002). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Jacquet BV, Salinas-Mondragon R, Liang H, Therit B, Buie JD, Dykstra M, Campbell K, Ostrowski LE, Brody SL, Ghashghaei HT, FoxJ1-dependent gene expression is required for differentiation of radial glia into ependymal cells and a subset of astrocytes in the postnatal brain. Development 136, 4021–4031 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Liang H, Hippenmeyer S, Ghashghaei HT, A Nestin-cre transgenic mouse is insufficient for recombination in early embryonic neural progenitors. Biol. Open 1, 1200–1203 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng H, A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat. Neurosci. 13, 133–140 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Mirzadeh Z, Merkle FT, Soriano-Navarro M, Garcia-Verdugo JM, Alvarez-Buylla A, Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain. Cell Stem Cell 3, 265–278 (2008). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Shah PT, Stratton JA, Stykel MG, Abbasi S, Sharma S, Mayr KA, Koblinger K, Whelan PJ, Biernaskie J, Single-cell transcriptomics and fate mapping of ependymal cells reveals an absence of neural stem cell function. Cell 173, 1045–1057.e9 (2018). [DOI] [PubMed] [Google Scholar]
  • 35.Imayoshi I, Sakamoto M, Kageyama R, Genetic methods to identify and manipulate newly born neurons in the adult brain. Front. Neurosci. 5, 64 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Kriegstein A, Alvarez-Buylla A, The glial nature of embryonic and adult neural stem cells. Annu. Rev. Neurosci. 32, 149–184 (2009). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Ellis P, Fagan BM, Magness ST, Hutton S, Taranova O, Hayashi S, McMahon A, Rao M, Pevny L, SOX2, a persistent marker for multipotential neural stem cells derived from embryonic stem cells, the embryo or the adult. Dev. Neurosci. 26, 148–165 (2004). [DOI] [PubMed] [Google Scholar]
  • 38.Lagace DC, Whitman MC, Noonan MA, Ables JL, DeCarolis NA, Arguello AA, Donovan MH, Fischer SJ, Farnbauch LA, Beech RD, DiLeone RJ, Greer CA, Mandyam CD, Eisch AJ, Dynamic contribution of nestin-expressing stem cells to adult neurogenesis. J. Neurosci. 27, 12623–12629 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Nusse R, Clevers H, Wnt/β-catenin signaling, disease, and emerging therapeutic modalities. Cell 169, 985–999 (2017). [DOI] [PubMed] [Google Scholar]
  • 40.Schulte G, Bryja V, The Frizzled family of unconventional G-protein-coupled receptors. Trends Pharmacol. Sci. 28, 518–525 (2007). [DOI] [PubMed] [Google Scholar]
  • 41.Lein ES, Hawrylycz MJ, Ao N, Ayres M, Bensinger A, Bernard A, Boe AF, Boguski MS, Brockway KS, Byrnes EJ, Chen L, Chen L, Chen T-M, Chin MC, Chong J, Crook BE, Czaplinska A, Dang CN, Datta S, Dee NR, Desaki AL, Desta T, Diep E, Dolbeare TA, Donelan MJ, Dong H-W, Dougherty JG, Duncan BJ, Ebbert AJ, Eichele G, Estin LK, Faber C, Facer BA, Fields R, Fischer SR, Fliss TP, Frensley C, Gates SN, Glattfelder KJ, Halverson KR, Hart MR, Hohmann JG, Howell MP, Jeung DP, Johnson RA, Karr PT, Kawal R, Kidney JM, Knapik RH, Kuan CL, Lake JH, Laramee AR, Larsen KD, Lau C, Lemon TA, Liang AJ, Liu Y, Luong LT, Michaels J, Morgan JJ, Morgan RJ, Mortrud MT, Mosqueda NF, Ng LL, Ng R, Orta GJ, Overly CC, Pak TH, Parry SE, Pathak SD, Pearson OC, Puchalski RB, Riley ZL, Rockett HR, Rowland SA, Royall JJ, Ruiz MJ, Sarno NR, Schaffnit K, Shapovalova NV, Sivisay T, Slaughterbeck CR, Smith SC, Smith KA, Smith BI, Sodt AJ, Stewart NN, Stumpf K-R, Sunkin SM, Sutram M, Tam A, Teemer BD, Thaller C, Thompson CL, Varnam LR, Visel A, Whitlock RM, Wohnoutka PE, Wolkey CK, Wong VY, Wood M, Yaylaoglu MB, Young RC, Youngstrom BL, Yuan XF, Zhang B, Zwingman TA, Jones AR, Genome-wide atlas of gene expression in the adult mouse brain. Nature 445, 168–176 (2007). [DOI] [PubMed] [Google Scholar]
  • 42.Stuebner S, Faus-Kessler T, Fischer T, Wurst W, Prakash N, Fzd3 and Fzd6 deficiency results in a severe midbrain morphogenesis defect. Dev. Dyn. 239, 246–260 (2010). [DOI] [PubMed] [Google Scholar]
  • 43.Wang Y, Thekdi N, Smallwood PM, Macke JP, Nathans J, Frizzled-3 is required for the development of major fiber tracts in the rostral CNS. J. Neurosci. 22, 8563–8573 (2002). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Proffitt KD, Madan B, Ke Z, Pendharkar V, Ding L, Lee MA, Hannoush RN, Virshup DM, Pharmacological inhibition of the Wnt acyltransferase PORCN prevents growth of WNT-driven mammary cancer. Cancer Res. 73, 502–507 (2013). [DOI] [PubMed] [Google Scholar]
  • 45.Huang DW, Sherman BT, Lempicki RA, Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009). [DOI] [PubMed] [Google Scholar]
  • 46.Supek F, Bošnjak M, Škunca N, Šmuc T, REVIGO summarizes and visualizes long lists of gene ontology terms. PLOS ONE 6, e21800 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Feldner A, Adam MG, Tetzlaff F, Moll I, Komljenovic D, Sahm F, Bäuerle T, Ishikawa H, Schroten H, Korff T, Hofmann I, Wolburg H, von Deimling A, Fischer A, Loss of Mpdz impairs ependymal cell integrity leading to perinatal-onset hydrocephalus in mice. EMBO Mol. Med. 9, 890–905 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Takagishi M, Sawada M, Ohata S, Asai N, Enomoto A, Takahashi K, Weng L, Ushida K, Ara H, Matsui S, Kaibuchi K, Sawamoto K, Takahashi M, Daple coordinates planar polarized microtubule dynamics in ependymal cells and contributes to hydrocephalus. Cell Rep. 20, 960–972 (2017). [DOI] [PubMed] [Google Scholar]
  • 49.Treat AC, Wheeler DS, Stolz DB, Tsang M, Friedman PA, Romero G, The PDZ Protein Na+/H+ Exchanger Regulatory Factor-1 (NHERF1) Regulates Planar Cell Polarity and Motile Cilia Organization. PLOS ONE 11, e0153144 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Katayama Y, Nishiyama M, Shoji H, Ohkawa Y, Kawamura A, Sato T, Suyama M, Takumi T, Miyakawa T, Nakayama KI, CHD8 haploinsufficiency results in autistic-like phenotypes in mice. Nature 537, 675–679 (2016). [DOI] [PubMed] [Google Scholar]
  • 51.Mariani J, Coppola G, Zhang P, Abyzov A, Provini L, Tomasini L, Amenduni M, Szekely A, Palejev D, Wilson M, Gerstein M, Grigorenko EL, Chawarska K, Pelphrey KA, Howe JR, Vaccarino FM, FOXG1-dependent dysregulation of GABA/Glutamate neuron differentiation in autism spectrum disorders. Cell 162, 375–390 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Won H, Mah W, Kim E, Kim J-W, Hahm E-K, Kim M-H, Cho S, Kim J, Jang H, Cho S-C, Kim B-N, Shin M-S, Seo J, Jeong J, Choi S-Y, Kim D, Kang C, Kim E, GIT1 is associated with ADHD in humans and ADHD-like behaviors in mice. Nat. Med. 17, 566–572 (2011). [DOI] [PubMed] [Google Scholar]
  • 53.Montarolo F, Martire S, Perga S, Spadaro M, Brescia I, Allegra S, De Francia S, Bertolotto A, NURR1 deficiency is associated to ADHD-like phenotypes in mice. Transl. Psychiatry 9, 207 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Molosh AI, Johnson PL, Spence JP, Arendt D, Federici LM, Bernabe C, Janasik SP, Segu ZM, Khanna R, Goswami C, Zhu W, Park S-J, Li L, Mechref YS, Clapp DW, Shekhar A, Social learning and amygdala disruptions in Nf1 mice are rescued by blocking p21-activated kinase. Nat. Neurosci. 17, 1583–1590 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 55.Okuda K, Takao K, Watanabe A, Miyakawa T, Mizuguchi M, Tanaka T, Comprehensive behavioral analysis of the Cdkl5 knockout mice revealed significant enhancement in anxiety- and fear-related behaviors and impairment in both acquisition and long-term retention of spatial reference memory. PLOS ONE 13, e0196587 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Furutachi S, Miya H, Watanabe T, Kawai H, Yamasaki N, Harada Y, Imayoshi I, Nelson M, Nakayama KI, Hirabayashi Y, Gotoh Y, Slowly dividing neural progenitors are an embryonic origin of adult neural stem cells. Nat. Neurosci. 18, 657–665 (2015). [DOI] [PubMed] [Google Scholar]
  • 57.Chaker Z, Codega P, Doetsch F, A mosaic world: Puzzles revealed by adult neural stem cell heterogeneity. Wiley Interdiscip. Rev. Dev. Biol. 5, 640–658 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Lupo G, Gioia R, Nisi PS, Biagioni S, Cacci E, Molecular mechanisms of neurogenic aging in the adult mouse subventricular zone. J. Exp. Neurosci. 13, 1179069519829040 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Li WL, Chu MW, Wu A, Suzuki Y, Imayoshi I, Komiyama T, Adult-born neurons facilitate olfactory bulb pattern separation during task engagement. eLife 7, e33006 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Silva-Vargas V, Maldonado-Soto AR, Mizrak D, Codega P, Doetsch F, Age-dependent niche signals from the choroid plexus regulate adult neural stem cells. Cell Stem Cell 19, 643–652 (2016). [DOI] [PubMed] [Google Scholar]
  • 61.Wang Y, Zhang J, Mori S, Nathans J, Axonal growth and guidance defects in Frizzled3 knock-out mice: A comparison of diffusion tensor magnetic resonance imaging, neurofilament staining, and genetically directed cell labeling. J. Neurosci. 26, 355–364 (2006). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Lehtinen MK, Zappaterra MW, Chen X, Yang YJ, Hill AD, Lun M, Maynard T, Gonzalez D, Kim S, Ye P, D’Ercole AJ, Wong ET, LaMantia AS, Walsh CA, The cerebrospinal fluid provides a proliferative niche for neural progenitor cells. Neuron 69, 893–905 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Johansson PA, Irmler M, Acampora D, Beckers J, Simeone A, Götz M, The transcription factor Otx2 regulates choroid plexus development and function. Development 140, 1055–1066 (2013). [DOI] [PubMed] [Google Scholar]
  • 64.Yang J, Brown A, Ellisor D, Paul E, Hagan N, Zervas M, Dynamic temporal requirement of Wnt1 in midbrain dopamine neuron development. Development 140, 1342–1352 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG, Improving bioscience research reporting: The ARRIVE guidelines for reporting animal research. PLOS Biol. 8, e1000412 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Mignone JL, Kukekov V, Chiang A-S, Steindler D, Enikolopov G, Neural stem and progenitor cells in nestin-GFP transgenic mice. J. Comp. Neurol. 469, 311–324 (2004). [DOI] [PubMed] [Google Scholar]
  • 67.Yamaguchi M, Saito H, Suzuki M, Mori K, Visualization of neurogenesis in the central nervous system using nestin promoter-GFP transgenic mice. Neuroreport 11, 1991–1996 (2000). [DOI] [PubMed] [Google Scholar]
  • 68.Zhang Y, Huang G, Shornick LP, Roswit WT, Shipley JM, Brody SL, Holtzman MJ, A Transgenic FOXJ1-cre system for gene inactivation in ciliated epithelial cells. Am. J. Respir. Cell Mol. Biol. 36, 515–519 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Yamaguchi M, Analysis of neurogenesis using transgenic mice expressing GFP with nestin gene regulatory regions. Chem. Senses 30, i117–i118 (2005). [DOI] [PubMed] [Google Scholar]
  • 70.Iwasato T, Nomura R, Ando R, Ikeda T, Tanaka M, Itohara S, Dorsal telencephalon-specific expression of Cre recombinase in PAC transgenic mice. Genesis 38, 130–138 (2004). [DOI] [PubMed] [Google Scholar]
  • 71.Matsumura H, Hasuwa H, Inoue N, Ikawa M, Okabe M, Lineage-specific cell disruption in living mice by Cre-mediated expression of diphtheria toxin A chain. Biochem. Biophys. Res. Commun. 321, 275–279 (2004). [DOI] [PubMed] [Google Scholar]
  • 72.Weichenhan D, Kunze B, Winking H, van Geel M, Osoegawa K, de Jong PJ, Traut W, Source and component genes of a 6–200 Mb gene cluster in the house mouse. Mamm. Genome 12, 590–594 (2001). [DOI] [PubMed] [Google Scholar]
  • 73.Shinmyo Y, Asrafuzzaman Riyadh M, Ahmed G, Bin Naser I, Hossain M, Takebayashi H, Kawasaki H, Ohta K, Tanaka H, Draxin from neocortical neurons controls the guidance of thalamocortical projections into the neocortex. Nat. Commun. 6, 10232 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 74.Yamauchi K, Yamazaki M, Abe M, Sakimura K, Lickert H, Kawasaki T, Murakami F, Hirata T, Netrin-1 derived from the ventricular zone, but not the floor plate, directs hindbrain commissural axons to the ventral midline. Sci. Rep. 7, 11992 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 75.Niwa H, Yamamura K, Miyazaki J, Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199 (1991). [DOI] [PubMed] [Google Scholar]
  • 76.Casali N, Preston A, E. coli Plasmid Vectors: Methods and Applications (Humana Press, 2003). [Google Scholar]
  • 77.Kelley LA, Mezulis S, Yates CM, Wass MN, Sternberg MJE, The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc. 10, 845–858 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 78.Ahsan M, Ohta K, Kuriyama S, Tanaka H, Novel soluble molecule, Akhirin, is expressed in the embryonic chick eyes and exhibits heterophilic cell-adhesion activity. Dev. Dyn. 233, 95–104 (2005). [DOI] [PubMed] [Google Scholar]
  • 79.Abdulhaleem FA, Song X, Kawano R, Uezono N, Ito A, Ahmed G, Hossain M, Nakashima K, Tanaka H, Ohta K, Akhirin regulates the proliferation and differentiation of neural stem cells in intact and injured mouse spinal cord. Dev. Neurobiol. 75, 494–504 (2015). [DOI] [PubMed] [Google Scholar]
  • 80.Sakuma T, Nakade S, Sakane Y, Suzuki K-IT, Yamamoto T, MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with the PITCh systems. Nat. Protoc. 11, 118–133 (2016). [DOI] [PubMed] [Google Scholar]
  • 81.Srinivas S, Watanabe T, Lin CS, William CM, Tanabe Y, Jessell TM, Costantini F, Cre reporter strains produced by targeted insertion of EYFP and ECFP into the ROSA26 locus. BMC Dev. Biol. 1, 4 (2001). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 82.Shoji H, Takao K, Hattori S, Miyakawa T, Age-related changes in behavior in C57BL/6J mice from young adulthood to middle age. Mol. Brain 9, 11 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 83.Ageta-Ishihara N, Yamazaki M, Konno K, Nakayama H, Abe M, Hashimoto K, Nishioka T, Kaibuchi K, Hattori S, Miyakawa T, Tanaka K, Huda F, Hirai H, Hashimoto K, Watanabe M, Sakimura K, Kinoshita M, A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance. Nat. Commun. 6, 10090 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 84.Hattori S, Takao K, Funakoshi H, Miyakawa T, Comprehensive behavioral analysis of tryptophan 2,3-dioxygenase (Tdo2) knockout mice. Neuropsychopharmacol. Rep. 38, 52–60 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 85.Kazdoba TM, Leach PT, Crawley JN, Behavioral phenotypes of genetic mouse models of autism. Genes Brain Behav. 15, 7–26 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 86.Mirzadeh Z, Doetsch F, Sawamoto K, Wichterle H, Alvarez-Buylla A, The subventricular zone en-face: Wholemount staining and ependymal flow. J. Vis. Exp., e1938 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 87.Park R, Moon UY, Park JY, Hughes LJ, Johnson RL, Cho S-H, Kim S, Yap is required for ependymal integrity and is suppressed in LPA-induced hydrocephalus. Nat. Commun. 7, 10329 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 88.Hatakeyama J, Bessho Y, Katoh K, Ookawara S, Fujioka M, Guillemot F, Kageyama R, Hes genes regulate size, shape and histogenesis of the nervous system by control of the timing of neural stem cell differentiation. Development 131, 5539–5550 (2004). [DOI] [PubMed] [Google Scholar]
  • 89.1000 Genomes Project Consortium, A global reference for human genetic variation. Nature 526, 68–74 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 90.Higasa K, Miyake N, Yoshimura J, Okamura K, Niihori T, Saitsu H, Doi K, Shimizu M, Nakabayashi K, Aoki Y, Tsurusaki Y, Morishita S, Kawaguchi T, Migita O, Nakayama K, Nakashima M, Mitsui J, Narahara M, Hayashi K, Funayama R, Yamaguchi D, Ishiura H, Ko W-Y, Hata K, Nagashima T, Yamada R, Matsubara Y, Umezawa A, Tsuji S, Matsumoto N, Matsuda F, Human genetic variation database, a reference database of genetic variations in the Japanese population. J. Hum. Genet. 61, 547–553 (2016). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 91.Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, Kondrashov AS, Sunyaev SR, A method and server for predicting damaging missense mutations. Nat. Methods 7, 248–249 (2010). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 92.Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano TK, Hodel MR, Petersen JF, Wyatt PW, Steenblock ER, Shah PH, Bousse LJ, Troup CB, Mellen JC, Wittmann DK, Erndt NG, Cauley TH, Koehler RT, So AP, Dube S, Rose KA, Montesclaros L, Wang S, Stumbo DP, Hodges SP, Romine S, Milanovich FP, White HE, Regan JF, Karlin-Neumann GA, Hindson CM, Saxonov S, Colston BW, High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal. Chem. 83, 8604–8610 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 93.Babraham Bioinformatics - FastQC A Quality Control tool for High Throughput Sequence Data; www.bioinformatics.babraham.ac.uk/projects/fastqc/.
  • 94.Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR, STAR: Ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 95.Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L, Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 96.Kim J-Y, Grunke SD, Levites Y, Golde TE, Jankowsky JL, Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction. J. Vis. Exp., 51863 (2014). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 97.Cheng Y, Phoon YP, Jin X, Chong SYS, Ip JCY, Wong BWY, Lung ML, Wnt-C59 arrests stemness and suppresses growth of nasopharyngeal carcinoma in mice by inhibiting the Wnt pathway in the tumor microenvironment. Oncotarget 6, 14428–14439 (2015). [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Direction of fluid flow generated by the ependymal cilia in WT mice and TSK-KO mice.

Movie S1. Direction of fluid flow generated by the ependymal cilia in WT mice and TSK-KO mice.

Download video file (14.8MB, mp4)
RNA-seq WT vs TSK-KO
NIHMS2188018-supplement-RNA-seq_WT_vs_TSK-KO.xlsx (1.4MB, xlsx)
Supplemental Material

Fig. S1. Behavioral test battery: Global behavior.

Fig. S2. Behavioral test battery: Three-chamber sociability and social novelty test (Crawley version).

Fig. S3. β-gal staining and TUNEL assay in RMS.

Fig. S4. IHC of the LV in TSK-hetero (TSK+/β-geo) mice.

Fig. S5. Relative expression of TSK protein in 10 μl of CSF (6.2 mg/ml) of WT and TSK-KO mice using anti-TSK antibody Mab1D3F1.

Fig. S6. Human TSK identified from patients with congenital hydrocephalus.

Fig. S7. Wnt family expression in mice.

Fig. S8. Cilia polarity.

Fig. S9. Coprecipitation assay.

Fig. S10. Summary of this study.

Table S1. Amplicon sequencing results of 11 single nucleotide variants derived from 13 patients by TSK, Fzd3, Fzd4, and BMP2B primers.

Table S2. Sanger sequencing of DNA samples from patients with hydrocephalus.

Table S3. Expression profiles of WT and TSK-KO mice isolated LV tissue by RNA-seq.

Table S4. The alterations of Wnt signaling genes in TSK-KO mice from comparative RNA-seq.

Table S5. GO analysis of comparative transcriptome at LV.

NIHMS2188018-supplement-Supplemental_Material.pdf (2.2MB, pdf)

Data Availability Statement

All data associated with this study are present in the paper or the Supplementary Materials.

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